Sertoli cells were collected from the testes of 21 day old, sexually immature Wistar rats. The cells were then incubated for 10 or 14 days in culture medium with or without the addition of FSH. This time period corresponds to the time period in vivo when rat Sertoli cells undergo active differentiation with concomitant histochemical and morphological changes. Cells cultured 10 and 14 days after initial plating were processed for the histochemical detection of three esterases and four dehydrogenases by observing relative staining intensities of azo dye precipitation and formazan reaction product, respectively. Appropriate controls were established. The presence of FSH mildly increased the staining activity of LDH, SDH and G-6-PDH in both 10 and 14 day cultured cells. However, SDH staining intensity/cell did not increase to surpass LDH staining intensity/cell as it does in vivo during this period of time. Addition of FSH also slightly increased staining of non-specific esterase. Type B esterase and 3 beta-ol DH activity was not evident in 10 and 14 day cultured cells, even in the presence of FSH. Our results indicate that, based on histochemical parameters, immature Sertoli cells do not mature in culture commensurate with the cell in vivo.