Monitoring the maturation of fish gonads requires an understanding of the locations of reproductive hormone formation both under the influence of environmental factors and under normal conditions. A crucial role in the biosynthesis of active steroid hormones is played by 3β-hydroxysteroid dehydrogenase (3β-HSD). The male and female Oreochromis niloticus gonads at different stages of the annual sexual cycle were analyzed for 3β-HSD using histochemical techniques to pinpoint the locations of steroid synthesis. In females, a specific cell type in the thecal layer, interstitial cells, and ovarian wall epithelial cells exhibited 3β-HSD activity. In males, only Leydig cells (interstitial cells) and lobule boundary cells exhibited 3β-HSD activity.  During the stages of sexual maturation and spawning, the Leydig cells' steroidogenic activity increased. Additionally, during the maturing and spawning stages, 3β-HSD exhibited a strong reaction that increased in intensity and the apparent quantity of cells exhibiting a reaction. However, after spawning, the reaction decreased. Furthermore, during the oogenesis process, which is the development of the previtellogenic oocytes to the ripe oocytes, 3β-HSD activity was elevated in the various phases of ovarian maturation. In conclusion, it can be stated that the cells producing steroid hormones in the male and female gonads of O. niloticus have been sufficiently identified, and their steroid hormone-producing capacity has been demonstrated. Naturally, more research is necessary to apply this finding to the development of fish gonads during the reproduction in aquaculture settings or under situations where this teleost species is concerned to environmental treatments.