17-beta-Estradiol dehydrogenase is required for the enzymatic interconversion of estradiol and its weaker related sex steroid, estrone. We isolated and sequenced a complementary deoxyribonucleic acid clone for 17 beta-estradiol dehydrogenase from the BeWo choriocarcinoma cell line. Comparison of the BeWo complementary deoxyribonucleic acid sequence to a previously derived placental complementary deoxyribonucleic acid sequence yields greater than 98% homology. We also isolated the gene for 17 beta-estradiol dehydrogenase from the Jar human choriocarcinoma cell line and elucidated its primary nucleic acid structure. Significant differences in the Jar-deduced complementary deoxyribonucleic acid sequence clearly differentiate it from both the human placental and BeWo forms of 17 beta-estradiol dehydrogenase, indicating the existence of two genes for 17 beta-estradiol dehydrogenase in the human genome. Evaluation of 17 beta-estradiol dehydrogenase gene expression in BeWo and Jar cells was compared with expression in luteinized granulosa cells. Messenger ribonucleic acid for human placental 17 beta-estradiol dehydrogenase was identified in all three cell types as a 1.3 kilobase band on Northern blot analysis. A second messenger ribonucleic acid species measuring 2.1 kilobase was abundantly present in the granulosa cells. Whether these two species of messenger ribonucleic acid are involved in the regulation of the estradiol dehydrogenase genes is yet to be determined.