OBJECTIVE Platelet-activating factor (PAF) activates p38, an important intracellular signal transduction kinase, and primes human mononuclear cells for the production of interleukin 8 (IL-8), a potent chemoattractant and activator of neutrophils. METHODS Human mononuclear cells were isolated from healthy adults by Ficoll-paque density-gradient centrifugation. Interleukin-8 in the supernatant was measured by enzyme-linked immunosorbent assay. Dual phospho-specific p38 antibody was used to detect activated p38 by Western blotting. RESULTS Lipopolysaccharide (LPS) and PAF activated p38. There was a shorter latency to peak p38 activation with PAF vs LPS stimulation, 5 vs 30 minutes. Platelet-activating factor-induced p38 activation was calcium dependent because it was inhibited by ethyleneglycoltetracetic acid. Lipopolysaccharide, 0.01 to 1.00 ng/mL, induced significant IL-8 production. Although PAF did not induce significant IL-8 production, it potentiated LPS-induced IL-8 production. Production of IL-8, in response to LPS alone or in combination with PAF, was inhibited by SB202190, a specific p38 inhibitor. CONCLUSIONS Although LPS and PAF activated p38, only LPS induced IL-8 production; PAF acted as a priming agent. It seems that p38 activation is necessary but not sufficient for IL-8 production by human mononuclear cells. Identifying and evaluating the activation state of inflammatory signal transduction pathways might lead to methods for controlling and preventing neutrophil-induced tissue injury without interfering with the normal host immune response.