Adenylate cyclase activities were examined in mouse LM cell membranes which had been supplemented with polar head groups and/or fatty acids. Basal, fluoride-, and PGE1-stimulated activities varied systematically with changes in phospholipid composition, and PGE1-stimulated activities correlated with the average degree of unsaturation of the phospholipid fatty acids or with the primary amino group character of the phospholipid polar head groups. In addition, the Km for ATP of basal adenylate cyclase was systematically changed by both polar head group and fatty acid supplementation. Alteration of the membrane lipid composition also changed the temperature dependence of the enzyme and the lag time between addition of PGE1 and the onset of a change in catalytic rate. However, none of the alterations in the enzyme activity could be correlated with the viscosities of supplemented membranes and, instead, seemed to be characteristic for a specific polar head group or fatty acid composition. The data suggest a specific interaction of the enzyme with phospholipids and indicate that structural features of phospholipids may play a role in regulating adenylate cyclase activity. It is proposed that adenylate cyclase can exist in several different conformations in the membrane depending upon the phospholipid composition.