The role of phospholipase A2 (PLA2) and its metabolite arachidonic acid (AA) in the proliferation and differentiation of HL-60 cells was investigated. Addition of either 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or retinoic acid (RA) to HL-60 cells for 2 h inhibited PMA-stimulated PLA2 activity measured by [3H]AA release. The inhibitor of PLA2 activity, p-bromophenacyl bromide (BPB), significantly inhibited the proliferation of HL-60 cells and of fibroblast L929 and Swiss 3T3 cells in a dose-dependent manner. The effect of BPB on proliferation is probably through its inhibitory effect on PLA2 activity, since the same doses of BPB which inhibited proliferation also inhibited PLA2 activity determined by [3H]AA release. The importance of PLA2 activity for cell growth was further supported by the effect of two other PLA2 inhibitors, AACOCF3 and scalaradial, which inhibited HL-60 proliferation in a dose-dependent manner. BPB, AACOCF3 and scalaradial significantly increased the doubling time to 32.4 h, 34.0 h and 31.8 h, respectively, compared with 24.6 h in the control. The inhibitory effect of BPB on HL-60 proliferation was reversed by addition of exogenous free AA to HL-60 cells, indicating the importance of this metabolite for the proliferation process. This reversible effect is specific for AA since it was not achieved by other fatty acids like linolenic acid (LA) or oleic acid (OA). Addition of free AA to HL-60 cells did not induce differentiation, as expected. Although BPB, AACOCF3, or scalaradial inhibited proliferation, they did not induce differentiation nor affect the differentiation induced by 1,25(OH)2D3 or RA. These results implicate that PLA2 activity has no regulatory role in differentiation of HL-60 cells. The differential effect of PLA2 inhibitors on proliferation and differentiation of HL-60 cells suggests that these two processes function under different regulatory mechanisms.