Insulin lowers basal levels of tyrosinase activity and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. Insulin exerts its inhibitory effects in a typical dose-response manner, with maximal inhibition of enzyme activity occurring at 10-7 M. At maximal inhibition, tyrosinase activity is reduced to approximately 50% of the control levels. This inhibition precedes the observed inhibitory effect on cellular proliferation. Insulin not only lowers cell responsiveness to MSH, but also inhibits the tyrosinase stimulation produced by either theophylline or (Bu)2cAMP. Neither control levels nor MSH-mediated elevated cellular levels of cAMP were altered by insulin (10-7 M). These findings suggest that insulin exerts its inhibitory effects at a site distal to cAMP production. The inhibitory effect of insulin on tyrosinase activity could not be mimicked by either (Bu)2cGMP or 8-bromo-cGMP, suggesting that insulin does not exert its effects by altering cellular levels of this nucleotide. Insulin reduces the rate of incorporation of [3H]leucine into trichloroacetic acid-precipitable material by 50%, a finding which suggests that insulin may exert its inhibitory effects on tyrosinase activity and perhaps on cellular proliferation by causing a general reduction in protein synthetic rates.