The effects of hydrogen ion concentration on high-affinity agonist binding to muscarinic receptors were determined in rat brainstem membranes using [3H]oxotremorine-M as a probe. [3H]Oxotremorine-M bound with high affinity to a small subpopulation of brainstem muscarinic receptors (10% of the receptors labelled by [3H]methylscopolamine). [3H]Oxotremorine-M binding was constant between pH 7.0 and 9.0; the number of high-affinity sites decreased below pH 7.0 and at pH 5.0 no binding was detected. This decrease was irreversible; when brainstem membranes were incubated for 1 h at low pH and then returned to pH 8.0, agonist binding was not restored. In contrast, the total number of receptors, i.e. the number of [3H]antagonist binding sites, was not affected by prolonged incubation at low pH. Agonist affinity for the surviving [3H]oxotremorine binding sites and the sensitivity of agonist binding to guanine nucleotides were not altered in media of low pH (pH 5.5). These findings suggest that [3H]oxotremorine-M binds only to receptors which are functionally coupled to guanine nucleotide-dependent regulatory proteins and that this coupling is irreversibly inactivated in media of low pH.