The presence of steroid hormones in the aquatic environment is potentially threatening the population dynamics of many species of animals and public health. Environmental estrogens in water have been reported to be associated with abnormal sexual development and abnormal feminizing responses in some animals. New approaches for the bioremediation of steroid hormones from the environment are therefore urgently sought. The researchers at Kiel University have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. In the present investigation, 16S rRNA analysis showed that marine strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. Bacterial strain H5 can degrade steroids such as testosterone and estrogens, which was shown in this study by determining the 3H labeled steroid retaining in the bacterial H5 culture medium at incubation times of 5h and 20h. Since 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in steroid degradation in Comamonas testosteroni, a meta-genomic system with the 3α-HSD/CR gene as a positive control was established to enable identification of steroid inducible genes from bacterial strain H5. In this meta-genomic system, a library of H5 chromosomal DNA fragments cloned into a fluorescent reporter pKEGFP-2 was constructed. A reporter plasmid pK3α-4.6-EGFP3 containing the 3α-HSD/CR gene was created as a positive control. Steroid induction could be detected by a microplate fluorescence reader, when the plasmids were transformed into Escherichia coli HB101 cells. With this meta-genomic pKEGFP-2 approach, two estradiol-inducible genes were identified from marine strain H5, which are obviously involved in steroid degradation. Sequencing of the pKEGFP-2 inserts and database research at NCBI revealed that one gene corresponds to 3- ketosteroid-delta-1-dehydrogenase from several Mycobacterium strains, while the other showed high similarity to carboxylesterase in Sebadella termitidis and Brachyspira murdochii. Both 3-ketosteroid-delta- 1-dehydrogenase and carboxylesterase are one of the first enzymes in steroid degradation. The 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase genes were subcloned into plasmids pET38-12 and pET24-17, respectively. Overexpression in Escherichia coli strain BL21(DE3)pLysS cells resulted in corresponding proteins with an N-terminal His-tag sequence. After induction with Isopropyl-β-D-Thiogalactopyranoside, 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were purified in one-step using nickel-chelatechromatography. After protein determination, 3-ketosteroid -delta-1-dehydrogenase (0.48mg/ml) and carboxylesterase (1.28mg/ml) were used to prepare antibodies to determine steroid binding specificity in future research. In addition, a strain H5 specific DNA sequence of 480 bp was identified, which allows sensitive PCR detection, and quantification of strain H5 bacteria in “unknown” seawater samples. Currently, the exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged. This study has shown that the marine strain H5 could metabolize steroids; have isolated two estradiol inducible genes from strain H5 chromosomal DNA; purified the corresponding proteins for further research and built a new method to detect and quantify strain H5 by PCR approach. In summary, the strain H5 will play an important role for the bioremediation of steroid contaminations in seawater.