The localization of histamine receptors and their classes on the microvascular endothelium was assessed by using the electron-opaque conjugate histamine-ferritin (HF). In order to further check the specific binding of this conjugate, two compounds chemically similar to histamine but biologically inactive, tele-methylhistamine (t-MH) and 4-pyridylethylamine (PEA), and a specific H2 histamine receptor antagonist, SK&F 93479, were used. Glutaraldehyde-activated ferritin was covalently coupled with either histamine, as a biologically active HF conjugate, or with tele-methylhistamine, as a biologically inactive conjugate tele-methylhistamine-ferritin (t-MHF). The purity of the conjugates was determined by thin-layer chromatography. The HF conjugate (25 mg protein/100 g body weight) was perfused into RAP mice and bipolar microvascular fields of the diaphragm were fixed and processed for electron microscopy. The HF conjugate decorated restricted domains of the luminal aspect of the microvascular endothelium. The specific density of HF binding, recorded as the average number of binding sites per square micrometer of the luminal endothelial surface, was relatively high in venules (18.46 +/- 5.73) and lower in arterioles (12.89 +/- 6.13) and capillaries (9.51 +/- 4.20). The binding specificity of the HF conjugate was assessed through six groups of experiments: perfusion before HF conjugate with either histamine, tele-methylhistamine or 4-pyridylethylamine, perfusion with t-MHF conjugate only, or administration before t-MHF conjugate of either histamine or tele-methylhistamine. The statistical significance of the data was analyzed by using the "F" distribution test and the "Wilcoxon-Mann-Whitney rank-sum test". Both tele-methylhistamine and 4-pyridylethylamine as well as histamine, showed a higher competition on arterioles and venules than on capillaries. Neither tele-methylhistamine nor histamine inhibited the binding of t-MHF conjugate on the endothelium. Experiments using SK&F 93479 as a very potent H2 histamine receptor antagonist perfused before HF administration, suggested the existence of H2 histamine receptors at a higher concentration in venules (47.03%) and arterioles (38.80%) than in capillaries (14.16%). These findings demonstrate that HF conjugate binds specifically to the histamine receptors of microvascular endothelium, which are characteristically frequent and predominantly of H2 type in venules.