This report focuses on the identification of the human peripheral blood mononuclear cells that do or do not produce prostaglandins (PGs) and related arachidonic acid metabolites. Our results, using two different assay systems, indicate that the monocyte/macrophage (Mphi) is the major and possibly sole source of thromboxane (TXB2) and prostaglandin E2 (PGE2) among peripheral blood mononuclear cells. Adherent peripheral blood monocytes (> 95% esterase positive) produced substantial amounts of these compounds. Quantitation of products which had incorporated exogenous 14C-arachidonic acid and radioimmunoassay of adherent cell culture fluids demonstrated that the amount of TXB2 produced by these cells was appreciably greater than the amount of PGE2 produced. Additional confirmation of TXB2 synthesis was shown by abolishing the TXB2 peak on TLC and TXB2 activity detected by RIA by treating cells with a specific inhibitor of thromboxane synthetase. In contrast, non-adherent T cells failed to synthesize either PGE2 or TXB2. Non-adherent B cells (95% Ig positive) incubated with 14C-arachidonic acid produced a small peak of radioactivity co-chromatographing with TXB2, and no PGE. All three cell populations incorporated similar amounts of 14C-arachidonic acid into hydroxy-fatty acids. We were unable to detect 6-keto-F1 alpha, the hydrolysis product of prostacyclin (PGI2) in any of the cell types tested. The absence of PG synthesis among normal peripheral blood T and B cells was also noted among established human lymphoid cell lines. Neither a human T (CCRF), nor a human B-cell line (GM-130), produced PGE2 or TXB2. Three murine macrophage cell lines, P388D1, J774.2, and WEHI-3 produced PGE2 and the latter TXB2 as well.