Studies are reported on two different types of activated ketones as inhibitors of two important intracellular phospholipase A(2)s (PLA(2)) : the group IV 85 kDa Ca2+-dependent phospholipase A(2) (cPLA(2)) and the P388D(1) Ca2+-independent phospholipase A(2)(iPLA(2)). In a mixed micelle assay, we observed that the reaction progress curve of cPLA(2) in the presence of a trifluoromethyl ketone (TFMK) is linear at pH 7.4, while at pH 9.0 it is nonlinear and slows with time. An investigation of this discrepancy demonstrated that the TFMKs are slow, tight-binding inhibitors of the cPLA(2) at both pH’s, that the rate of dissociation of the enzyme-inhibitor complex is the same at both pH’s, but that the rate of association of enzyme and inhibitor is slower at pH 7.4 than at pH 9.0. A novel group of activated ketone inhibitors has been synthesized that contain a fatty acyl tricarbonyl. These compounds also inhibit the cPLA(2) in the mixed micelle assay. The inhibition of cPLA(2) by the tricarbonyls is readily reversible upon dilution and does not involve slow binding. For both types of inhibitor, no preference for fatty acid chain was observed as the palmityl analogs inhibited as well as the arachidonoyl analogs, despite the fact that the cPLA(2) shows a strong preference for arachidonoyl-containing phospholipid substrates over palmitoyl-containing substrates. With the iPLA(2), the inhibition by TFMKs is reversible and does not involve slow or tight binding. The tricarbonyls also inhibited the iPLA(2), but were less potent than the TFMKs. Unlike the cPLA(2), the iPLA(2) does exhibit a fatty acid preference as the palmityl analogs of both compounds inhibit better than the arachidonoyl analogs. The palmityl TFMK displayed a 10-fold lower IC50 at pH 9.0 than at pH 7.5, whereas the potency of the tricarbonyl was unchanged in this range. Thus, the TFMKs inhibit both the cPLA(2) and the iPLA(2), but the mechanism of inhibition of the two enzymes appears to be quite different. The tricarbonyls also inhibited both enzymes, but in both cases in a reversible manner and as such appear to be poorer inhibitors than the TFMKs.