Studies were performed to assess the effects of copper treatment in vitro on muscarinic binding parameters in rat brain homogenates. Brainstem, an area low in copper, was found to be insensitive to copper treatment as compared to forebrain, a region of relatively high copper content. Inclusion of 3 microM copper in forebrain homogenates decreased the number of sites seen by [3H]-l-quinuclidinyl benzilate (QNB) by 40-50%. Copper-enhanced displacement of bound QNB was noted for agonists and antagonists. Both ligands showed maximal effects at 6 microM copper, although quantitative differences could be determined at any copper level. At levels of maximal effect, the increase in QNB displacement was greater than or less than 50% for agonists and antagonists respectively. Two-site analyses of carbamylcholine (CCH) binding showed that the addition of 1 microM copper to forebrain homogenates increased the percentage of high affinity sites (alpha) from 42 to 70%. The IC50 decreased from 3.1 to 1.7 microM, but the dissociation constants for the high and low affinity sites were not changed. The effect of added copper on CCH binding to muscarinic receptors was reversible with the addition of the copper-chelating agent triethylene tetramine.