The chemotactic peptide fMLP (N-formyl-methionyl-leucyl-phenylalanine) induced the production of platelet-activating factor (PAF) by human polymorphonuclear leukocytes (PMN) incubated with cytochalasin B (CB). CoA-independent transacylase showed similar activity in both resting and stimulated PMN, and PAF production only occurred when lyso-PAF:acetyl-CoA acetyltransferase had been converted into the high activity form. PAF formation was coincidental with an increase of the concentration of cytosolic Ca2+ ([Ca2+]i), and with an enhanced formation of 1-O-[3H]alkyl-2-acyl-sn-glycerol. Both fMLP-induced PAF production and the activation of lyso-PAF:acetyl-CoA acetyltransferase were diminished by propranolol. Since several molecular species of phosphatidic acid (PA) produced an inhibition of both PAF production and acetyltransferase activation on intact cells, a portion of the inhibitory effect of propranolol was related to the accumulation of PA. Furthermore, whereas CB increased both the extent and the duration of the fMLP-induced [Ca2+]i transient, propranolol was found to inhibit the CB-induced increase of the [Ca2+]i transient. These data indicate that both the attenuation of [Ca2+]i transient and the accumulation of PA may operate as termination signals for PAF production by actin on lyso-PAF:acetyl-CoA acetyltransferase.