The correlation between membrane phospholipid composition and total cyclic AMP levels was investigated by using Neurospora lipid auxotrophs under various supplementation conditions. The lipid composition of the supplemented cultures was determined, and the intracellular and extracellular cyclic AMP levels were measured at various stages of the culture growth. Kinetic parameters and the thermostability of adenylate cyclase and of cyclic AMP-dependent phosphodiesterase were measured under all supplementation conditions. In inositol deficient inl cultures the levels of intracellular cyclic AMP decreased exponentially towards the end of the log phase and thereafter. In chol-l; chol-2 cultures, grown in N-monomethylethanolamine and low choline supplementation, the level of intracellular cyclic AMP decreased as function of decreasing exogenous choline supplement. Rates of cyclic AMP extrusion in all cultures were comparable on dry weight basis, and thus not affected by the mycelial lipid composition. Adenylate cyclase activity and thermostability decreased under those supplementation conditions resulting in reduction of cyclic AMP. Cyclic AMP-dependent phosphodiesterase was insensitive to phospholipid changes. Accordingly, it is suggested that specific perturbations in cellular phospholipid composition affect the membrane-bound adenylate cyclase and hence the cyclic AMP synthesis in vivo.