The muscarinic antagonist 1-[benzilic 4,4'-3H]-quinuclidinyl benzilate [3H]-QNB) bound to a single class of non-cooperative sites in calf cerebral cortex membranes (KD = 0.29 nM and Bmax = 1.06 pM/mg protein). Computer-assisted analysis of the shallow pirenzepine/[3H]-QNB competition binding curves indicated that 68% of these sites were of the M1-subtype and the remaining 32% of the M2 subtype. Respective Ki-values for pirenzepine were 27 nM and 1.14 microM. Binding characteristics of the antagonist atropine and of the agonist carbachol for M2 were evaluated by performing competition binding with 0.5 nM [3H]-QNB in the presence of 2 microM pirenzepine. The binding characteristics for the M1 receptors were obtained indirectly by subtracting the curve for M2 from the total curve, or directly by competition binding with 0.3 nM [3H]-pirenzepine. Atropine competition curves were steep for M1 and M2 and were not affected by 1 mM GTP nor by 1 mM N-ethylmaleimide. The carbachol competition curve was shallow for M2. The steep curves for M1 indicate that this receptor subclass was only composed of low agonist affinity sites. GTP, which caused a rightward shift and a steepening of the carbachol competition curve for M2, did not affect the curves for M1. N-ethylmaleimide provoked a leftward shift and a steepening of the carbachol competition curve for M2 and abolished GTP modulation. A leftward shift was also observed for M1, but of a smaller magnitude (i.e. 3-4-fold for M1 compared to 17-fold for M2). These data suggest that, in calf brain cortex, M1 and M2 receptors show different susceptibility towards GTP and N-ethylmaleimide modulation.