We examined the effect of sulfhydryl compounds on the regulation of adenylyl cyclase by calcium in mouse cerebrum membranes. Isoproterenol-stimulated adenylyl cyclase (IP-AC) activity in the membranes was increased by addition of the optimum concentrations of calcium/calmodulin. However, in the presence of 0.01-0.07 mM glutathione (GSH), calcium/calmodulin inhibited the activity. At high concentrations of GSH (1-10 mM), the IP-AC activity was stimulated by calcium/calmodulin to a greater extent than that in the control (no GSH). Cysteine at less than 1.7 mM induced a similar inhibition of the IP-AC activity, but dithiothreitol did not. The activity of IP-AC measured in the absence of calmodulin decreased when calcium levels were greater than 300 microM. GSH at 0.05 mM enhanced the calcium-dependent inhibition (22% inhibition by 200 microM calcium), while 10 mM GSH lowered it. Calmodulin itself had no significant effect on the IP-AC activity, irrespective of the concentrations of GSH involved. It caused a small increase in the IP-AC activity that had been reduced by the presence of calcium and GSH. These results indicate that the redox status of sulfhydryls in adenylyl cyclase plays an important role in the calcium-mediated regulation of the enzyme. The enzyme becomes much more sensitive to the calcium-dependent inhibition after partial reduction of the sulfhydryls via the particular mode of reactin.