We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg2+ (5.0-50 mM), while it activates the same enzyme in the presence of Ca2+ (0.1-1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg2+ and the inhibitory effect of RMI 12330 A in the presence of Ca2+.