Histamine is released from mast cells and basophils by either immunological or nonimmunological mechanisms. Histamine, which is the most potent short acting mediator released from these cells, exerts its diverse biological actions by binding to cell surface histamine receptors. We report the affinity purification of histamine receptor proteins from Triton X-100 solubilized peripheral human blood mononuclear cells which include lymphocytes and monocytes. Three different designs of histamine affinity columns were constructed; all three resulted in the same material being eluted. This consisted of bands which on SDS-PAGE after boiling and reduction had the following molecular weights: 193K, 84K, 58K, 48K, 37K, and 16K. The most abundant bands were of molecular weights 193K, 48K, and 16K, and these were disulfide bonded together to form a high molecular weight complex. (The 58K band was present in lower amounts than the others, and in only a few fractions. It had the same molecular weight as the dimeric form of histamine methyltransferase which is present in small amounts in mononuclear cells and may therefore have copurified.) The histamine binding proteins described in this report were purified by conventional affinity chromatography, rather than by an expression cloning approach which obviates the use of any protein chemistry. Consequently, we had the advantage of being able to verify the histamine binding specificity of our purified proteins directly and with several independent assays as follows. The histamine binding specificity of all three columns was established by specific elution with histamine, by preabsorption of crude cell extract with excess free histamine prior to column application, and by comparison with control columns. Independent determination of the binding specificity, using a radioreceptor dot blot assay, of the eluate containing only the 193K, 48K, and 16K disulfide-linked subunits confirmed that the purified material bound specifically to [3H]histamine and that a 300-500-fold degree of purification from tissue extract had been obtained. Following cell surface radioreceptor cross-linking of radiolabeled histamine to intact mononuclear cells, the 16K band was detected, indicating it to be the ligand-binding subunit for histamine. These same three proteins were purified from T lymphocyte and monocytoid cell lines, indicating that both lymphocyte and monocyte subsets of mononuclear cells express these proteins. The trimolecular structure, consisting of 193K, 48K, and 16K subunits appears to be a novel histamine cell surface receptor protein complex as the molecular weights of the 193K, 48K, and 16K bands bore no relation to the predicted molecular weights of the recently expression cloned H1 receptor gene derived from bovine adrenal and H2 receptor gene derived from human parietal cells; this may reflect receptor heterogeneity within a tissue, between tissues, and/or between species.