Cluster analysis at Postnatal Day 8-20 of putative androgen-regulated genes in mice with Sertoli cell-selective knockout of the androgen receptor (SCARKO) has pinpointed 3 genes (Spinlw1, Gpd1, Drd4) with an expression pattern strongly resembling that of Rhox5, the definitive Sertoli cell (SC) androgen-regulated gene. We used organotypic testis cultures from day 8 mice to study control of these genes by (anti)androgens and FSH. Testis morphology and androgen induction of the studied genes were preserved for 48 h. Preincubation with ketoconazole for 24 h to block endogenous androgen production, followed by 24 h incubation with the synthetic androgen R1881 resulted in 45-, 5-, 19- and 6-fold induction of mRNA levels of Rhox5, Spinlw1, Gpd1, and Drd4 respectively. However, noticeable differences in control of the studied genes were observed. Rhox5 and Spinlw1 were fully induced by R1881 in the continuous (48 h) presence of ketoconazole whereas only marginal effects were observed on expression of Gpd1 and Drd4. Similarly, FSH only marginally affected expression of Rhox5 and Spinlw1 whereas it markedly increased Gpd1 and Drd4 expression. Explant cultures of SCARKO testes confirmed the differential effects of FSH on the studied genes and, for Gpd1, showed that the effect did not depend on a functional androgen receptor in SC whereas this was essential for the effects of FSH on Drd4. In conclusion, organotypic cultures represent the first in vitro approach to preserve androgen responsiveness of putative SC expressed genes. This approach facilitates detailed analysis of their regulation in ways not possible in vivo.