Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2-3 fold) the specific binding of [3H]-QNB or [3H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30 degrees C. Magnesium, on the other hand, increased (2-3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased Kd (WP) while magnesium increased Kd (MIC) for [3H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on antagonist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4 degrees C (24 hr), the stimulatory effect of Gpp(NH)p on [3H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [3H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like Gi, Go) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.