The initial aim of the present study was to clarify the role of platelet-activating factor (PAF-acether) : acetylhydrolase in the catabolism of PAF-acether in cultured human vascular endothelial (HVE) cells stimulated by thrombin. When HVE cells were pretreated with 2mM phenylmethylsulfonyl fluoride (PMSF, a serine proteinase inhibitor), about 2 times more PAF-acether was produced in response to 2.5 U/ml thrombin stimulation. The enhancing effect of PMSF on the mediator production could be partially explained by the inhibition of PAF-acether degradation in HVE cells, since the acetylhydrolase activity was suppressed by 32.7% in PMSF-treated cells, However, the activity of lyso PAF-acether: acetyl CoA acetyltransferase in unstimulated or thrombin-stimulated cells was 11.0-26.9% higher in PMSF-treated cells, although thrombin caused similar activation of the enzyme (3.4-4.1 fold) in both control and PMSF-treated cells. Furthermore, PMSF pretreatment increased the content of lyso PAF-acether and thrombin-induced radioactivity release from HVE cells loaded wth [3H] arachidonic acid, suggesting a higher activity of phospholipase A2 in PMSF-treated cells. These results suggest that the enhancement of thrombin-induced PAF-acether formation in PMSF-treated HVE cells is not only due to the inhibition of the acetylhydrolase, but also due to the influences on the activities of the acetyltransferase and other enzymes such as phospholipase A2.