Changes in expression of Leydig cell 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase/C17-20 lyase (P450(17alpha)) messenger RNA (mRNA) during pubertal development have not been well characterized in the rat. In the present study, expression of 3betaHSD and P450(17alpha) were determined in frozen sections of testes of immature (days 21 and 28), pubertal (days 45 and 60) and adult (day 90) rats by in situ hybridization using digoxigenin-labeled riboprobes and quantified densitometrically. Measures of steroidogenesis in this study, 3betaHSD and P450(17alpha) enzyme activities per testis and plasma testosterone concentration, increased during pubertal development, peaking at 45-60 days of age. Expression of 3betaHSD protein, a marker for Leydig cell function, was abundantly immunolocalized to the interstitial compartment of the testis. Quantified densitometrically, the amount of 3betaHSD protein did not vary significantly during pubertal development. Transcripts of 3betaHSD and P450(17alpha) were expressed abundantly by clusters of immature Leydig cells in immature animals. However, in contrast to measures of steroidogenesis during pubertal development, mRNA of 3betaHSD and P450(17alpha) decreased to undetectable levels at the age of 45 and 60 days, respectively. The decline in mRNA of 3betaHSD and P450(17alpha) was confirmed by Northern analysis. Expression of 3betaHSD and P450(17alpha) transcripts rebounded in the adult at 90 days and were comparable to levels of expression observed in immature animals. These results show that during pubertal development the steady-state accumulation of mRNA of 3betaHSD and P450(17alpha) are not correlated with accumulation of 3betaHSD protein, enzyme activities of 3betaHSD and P450(17alpha), or testosterone secretion. Possible explanations of the depletion of transcripts during pubertal development include: specific inhibition of transcription, increased mRNA instability, or high translational activity.