We studied effects of various agents, including follicle-stimulating hormone (FSH), on changes in the level of asparagine synthetase in cultured rat Sertoli cells. FSH, dibutylyl cAMP (Bt2cAMP), and cAMP-increasing agents, such as forskolin and theophylline, increased enzyme activity within 24 h. Western blot analysis revealed the increase in cellular protein content of asparagine synthetase by these agents to a degree similar to that of the enhanced enzyme activity. Northern blot analysis also showed elevation of the level of mRNA for asparagine synthetase in FSH- and Bt2cAMP-treated cells. Testosterone, insulin, prostaglandin E2, or 12-O-tetradecanoyl phorbol-13-acetate did not stimulate the enzyme. The results suggest that asparagine synthetase is induced via a cAMP-dependent signal transduction pathway in Sertoli cells. Increased production of asparagine in FSH- and Bt2cAMP-treated cells was indicated by extracellular accumulation of more asparagine around the treated cells than around the controls. The basal level of asparagine synthetase assessed in extracts from whole testes increased with age of animals toward 60-70 days, but the degree of hormonal stimulation of the enzyme in isolated Sertoli cells decreased during Days 20 to 30 postpartum. This suggested some different mechanism for regulation of the enzyme. The enhanced activity of asparagine synthetase by FSH in Sertoli cell may be important for maturation and function of Sertoli cells.