The thermodynamic properties of pirenzepine (PZ) binding to membrane-bound and digitonin-solubilized muscarinic receptors (mAChR) from the rat forebrain and heart were evaluated. Apparent dissociation constants (Kd) of PZ were measured from saturation studies using [3H]PZ for forebrain membrane-bound mAChR and from inhibition studies of (-)-[3H]quinuclidinyl benzilate binding using unlabeled PZ, at five different temperatures from 4 degrees C to 37 degrees C. The Kd values of PZ binding to both membrane-bound and solubilized mAChR decreased with decreasing temperature whereas the maximum receptor density was unchanged. The heterogeneity of membrane-bound mAChR characterized by PZ binding to mAChR from both tissues disappeared upon digitonin-solubilization of the mAChR. The magnitude of changes of the Kd values with temperature was greater in the solubilized mAChR, suggesting that some constituents in the membrane constrained the affinity changes. The Gibbs free energy of PZ binding to membrane-bound and solubilized mAChR were both negative. The Gibbs free energy for membrane-bound receptors decreased (more negative) whereas those for solubilized receptors increased (less negative) with increasing temperature. The change in entropy was the apparent major driving force for PZ binding to membrane-bound receptors with the change in enthalpy also being favorable. The change in enthalpy was the apparent major driving force for PZ binding to solubilized receptors at all temperatures with the change in entropy being unfavorable above 17 degrees C in the rat forebrain mAChR and above 10 degrees C in the heart mAChR. Our results suggest an important role for the biomembrane microenvironment and possible topographical differences in the binding sites which may contribute to the mechanism of muscarinic subtypes.