³H-Cimetidine binding to plasma membranes of isolated guinea pig gastric glands was investigated, and the effects of five H₂-receptor antagonists on ³H-cimetidine binding and histamine stimulation of cellular cAMP were compared. Of the five cations tested, Cu⁺⁺ markedly increased specific ³H-cimetidine binding. ³H-Cimetidine had high affinity (K_d=0.41×10^-6M) and low affinity (K_d=12.8×10^-6M) binding sites. Cimetidine and etintidine were potent inhibitors of ³H-cimetidine binding, while famotidine, ranitidine and TZU-0460 were not. Histamine stimulation of cellular cAMP was competitively inhibited by H₂-receptor antagonists yielding pA₂ values of 6.41 for cimetidine, 6.82 for etintidine, 6.87 for ranitidine, 6.94 for TZU-0460 and 7.60 for famotidine. Because the K_B value (log K_B=-pA₂) of 0.39×10^-6M for cimetidine is close to the K_d value for the high affinity ³H-cimetidine binding site, it is presumed to represent a part of the H₂-receptor, and the relative potency of etintidine against cimetidine in inhibiting ³H-cimetidine binding is similar to that in inhibiting histamine stimulation of cellular cAMP. These results suggest that imidazole-derived H₂-receptor antagonists (cimetidine and etintidine) and non-imidazole H₂-receptor antagonists (famotidine, ranitidine and TZU-0460) compete with histamine at different sites on the H₂-receptor of the gastric glands.