We developed an HPLC method utilizing fluorescence precolumn labeling to determine the concentration of a series of eicosanoids. LTB4 and cyclooxygenase products of arachidonic acid were derivatized with 9-anthryldiazomethane as previously reported and detected by their fluorescence in the HPLC eluate. The value measured by this method for LTB4 showed a good correlation with that obtained by RIA, as was the case for previously reported cyclooxygenase products. By this method LTB4, 6-keto-PGF1 alpha, and PGE2 were detected in the incubation medium of rat leukocytes. The leukocytes, which contained approximately 90% PMNL, were collected after intraperitoneal injection of casein, and were stimulated with Ca-ionophore A23187 or platelet-activating factor (PAF) in vitro. Release of LTB4 from 4 x 10(7) cells showed peaks of 450, 50, 23 ng at 5 min after stimulation with Ca-ionophore A23187 (12.5 microM), C16-PAF (1.0 microgram/ml), and C18-PAF (1.0 microgram/ml), respectively. The amount of 6-keto-PGF1 alpha continued to increase up to 20-40 min of incubation by those stimulators and showed respective maximum values of 100, 40, and 20 ng. The result suggests that inflammatory mediators, such as PAF, can act on inflammatory cells to release second mediators that act synergistically at the site of inflammation.