1. The binding of (-) [3H]quinuclidinylbenzilate (QNB), a potent muscarinic antagonist, to cardiac muscarinic receptors was examined in washed particles and microsomes isolated from rat heart atria. 2. Addition of divalent cations (Mg2+, Mn2+ and Ca2+) to the binding assays increased (6 to 8-fold) the antagonist binding to microsomes but exerted little or only weak (up to 35%) stimulation of the antagonist binding to washed particles. 3. Mg2+ ions present only in the dilution and washing buffer prior to rapid filtration of the binding reaction were capable of markedly increasing the antagonist binding to microsomes but not to washed particles. 4. Such differential action of Mg2+ on antagonist binding to subcellular fractions was seen when fractions were either isolated and or incubated in media containing either 10 or 50 mM imidazole-Cl, Tris-phosphate or Tris-Cl buffer, each at pH 7.5. While the modulatory effect of Mg2+ was not seen in Na-K-phosphate buffer, pH 7.5, the antagonist binding per se was much higher in Na-K-phosphate buffer compared to other buffers.