Human leukocytes generate platelet-activating factor (PAF-acether), a lipid mediator of inflammation, from membrane alkyl phospholipids through the release of arachidonic acid or other fatty acids at the 2-position and subsequent acetylation. Because it was previously demonstrated that fish oil fatty acids suppress human leukocyte arachidonic acid release and metabolism, separate experiments were conducted to investigate the effects of dietary fish oil supplementation and in vitro incubation with fish oil fatty acids on calcium ionophore-stimulated PAF-acether generation in human monocytes. In subjects on their regular diets, a 4-hr incubation of monocyte monolayers with an optimally effective concentration of arachidonic acid of 1 micrograms/ml resulted in a 64% increase of calcium ionophore-induced net PAF-acether generation from 7.75 +/- 0.78 ng/10(6) cells for untreated monolayers to 12.70 +/- 1.21 ng/10(6) cells (mean +/- SEM). Treatment of monolayers with eicosapentaenoic acid (EPA) at the optimal concentration of 1 micrograms/ml decreased net PAF-acether generation by 28%. However, treatment of monocyte monolayers with docosahexaenoic acid did not appreciably affect net PAF-acether generation. The changes in PAF-acether release with each fatty acid added in vitro paralleled those in total PAF-acether generation; the percentage PAF-acether release remained unaffected. Three weeks of dietary supplementation with 18 g MaxEPA daily, providing 3.2 g EPA did not affect the PAF-acether generation of calcium ionophore-stimulated human monocyte monolayers. However, 6 weeks of dietary supplementation resulted in a 47% decrease of net total PAF-acether generation and a concomitant 59% decline in net PAF-acether release; the percentage release of PAF-acether was not affected. Thus, whether added to the diet or introduced in vitro, fish oil-derived fatty acids suppress PAF-acether generation by human monocyte monolayers.