To assess the regulation of stem factor factor (SCF) gene expression during spermatogenesis, we tested the effects of hormones (FSH, testosterone, and 17β-estradiol) and some growth factors[ transforming growth factor-β (TGFβ), TGFα, tumor necrosis factor-α, and activin] on SCF gene expression by using a transillumination-assisted microdisection technique, a seminiferous tubule culture system, and Northern hybridization. Our results showed that FSH (10 ng/ml) increased steady state levels of SCF messenger RNA (mRNA) in a stage-specific and time-dependent manner. 8-Bromo-cAMP could increase the SCF mRNA level in a similar way as FSH, whereas phorbol 12-myristate 13-acetate had no effect. Actinomycin D could abolish the stimulatory effect of FSH, whereas cyclohexamide could not. The half-life of SCF mRNA was apparently prolonged after FSH stimulation (FSH-treated tubules, 15.6 ± 1.2 h; controls, 8.6 ± 2.7 h). Nuclear run-on assay revealed 5- and 10-fold increases in the transcription rate after FSH stimulation for 8 and 30 h, respectively. Neither testosterone nor estradiol had significant effects on SCF gene expression in our tissue culture system. Activin, TGFβ, TGFα, and tumor necrosis factor-α had no effect on SCF gene expression in vitro. In conclusion, SCF gene expression in the rat seminiferous tubule is regulated by FSH through the cAMP/protein kinase A pathway. FSH regulates SCF gene expression at both transcriptional and posttranscriptional levels involving the increase in transcription rate and prolongation of half-life of SCF mRNA, but is independent of de novo protein synthesis.