Leukotrienes (LTs) are potent mediators of inflammatory and allergic responses, and are present in biological fluids in minute amounts, that is, in the picogram range. The aim of this study was to develop and validate a method for determination of LTB₄ synthesized in vitro in human whole blood. Heparinized blood was stimulated with calcium-ionophore A23187 at 37°C. After 30 min cells were separated by centrifugation. LTB₄ was analyzed by radioimmunoassay (RIA). When sample preparation was restricted to protein precipitation with acetone, interference was demonstrated by lack of parallelism between standard and sample dilution curves. Purification was, therefore, extended by combinations of the following steps: 1) protein precipitation, 2) lipid extractions, and 3) high-performance liquid chromatography (HPLC). One of two commercially available LTB₄ standards was found to contain multiple components, several of which were immunoreactive in RIA. Even for the standard containing pure LTB₄, interference was demonstrated by lack of parallelism between sample and standard dilution curves. Testing eight combinations of varying purification steps, we found that only a three-step purification procedure, including 1) solid-phase extraction, 2) protein precipitation at -20°C, and 3) HPLC, was able to eliminate interference in RIA. Using this procedure, the recovery was 78%. Stimulation of whole blood from normal subjects with calcium-ionophore showed optimal LTB₄ production at 10 μM ionophore, yielding 6.6 ng LTB₄/mL blood.