A highly sensitive and rapid high-performance liquid chromatographic assay for the determination of histamine and 3-methylhistamine in biological samples using 1-methylhistamine as the internal standard is described. Samples were purified and concentrated on cation-exchange columns and derivatized with fluorescamine. The lower detection limit was 20 pg on-column. Linearity was demonstrated up to 20 ng on-column. The samples could be derivatized simultaneously before injection and were stable for 7 days. The method was used for the determination of histamine and related compounds in coronary perfusates, extracts of homogenized rat hearts, and supernatants of stimulated peritoneal mast cells.