The mode of the inhibition of purified rat brain adenylate cyclase by the beta gamma-subunits of G protein (beta gamma) was studied. These subunits inhibited the catalytic activity of the cyclase with the maximal inhibition of 85% and the half-maximal inhibition at about 0.7 nM beta gamma. The complex of beta gamma and adenylate cyclase isolated by density gradient centrifugation contained 1.8-2.0 mol beta gamma per mol of the cyclase when beta gamma was assayed by immunoblotting and by its inhibitory activity on adenylate cyclase. However, the beta gamma concentration-inhibition curves suggest that one of the two beta gamma molecules bound may be essential for the inhibition. The role for the second beta gamma molecule is unknown. As a tentative estimate, 70% of the adenylate cyclase activity remained inhibited by beta gamma when the complex was isolated. The inhibition was not dependent on G alpha s or calmodulin. Although purified adenylate cyclase contained a protein (0.06-0.08 mol/mol of adenylate cyclase) that reacted with anti-G alpha s antibody, this protein was not liberated from the cyclase when it formed a complex with beta gamma. In addition, guanine nucleotide analogs little affected the cyclase activity or the inhibition by beta gamma. The inhibition by beta gamma was reversed by the dilution of the complex, and the following re-addition of beta gamma suppressed the enzyme activity to about 15% of the initial activity again. These findings provide strong evidence that beta gamma inhibits adenylate cyclase directly and reversibly through the formation of the complex.