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Multiple forms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and their differential tissue expression pattern have not been shown in any lower vertebrates. In the present study, we report cloning of two novel 3β-HSDs and two variants from gonads of the Nile tilapia. 3β-HSD cDNAs encode two peptides of 375 (3β-HSD type-I/variant 1) and 367 (3β-HSD type-II/variant 1) amino acid residues that share 31.9% homology. 3β-HSD type-I/variant 1 shared high homology with other piscine counterparts while 3β-HSD type-II/variant 1 exhibited homology to mammalian ΔC27-3β-HSD and multifunctional viral 3β-HSD. The latter seems to be ancient form among vertebrates. Transiently transfected 3β-HSDs’ open reading frames in COS-7 cells converted exogenous pregnenolone/androsta-5-ene-3β-17β-diol to progesterone/testosterone. Tissue distribution pattern of 3β-HSDs by RT-PCR revealed varied …