Previous studies have shown that 1,25-(OH)2D3 stimulates phospholipase A2 (PA2) activity in growth zone chondrocytes (GC), but has no effect on the resting zone chondrocyte (RC) enzyme activity. 24,25-(OH)2D3 inhibits the RC enzyme but has no effect on the GC. This study examined whether the vitamin D metabolites affect arachidonic acid turnover in their contra-target cell populations. Incorporation and release of [14C]arachidonate was measured at various times following addition of hormone. Acylation and reacylation were measured independently by incubating with p-chloromercuribenzoate. The results demonstrated that 1,25-(OH)2D3 has no effect on arachidonic acid turnover in RC, but stimulates turnover in GC. In contrast, 24,25-(OH)2D3 stimulates arachidonic acid turnover in RC, but inhibits both incorporation and release in GC. To determine whether direct interaction with PA2 is one mechanism by which 1,25-(OH)2D3 and 24,25-(OH)2D3 regulate arachidonic acid release, snake venom (Niger niger) PA2 was incubated with the vitamin D metabolites. Enzyme specific activity was inhibited by 24,25-(OH)2D3 and stimulated by 1,25-(OH)2D3 in a time- and dose-dependent manner. These results suggest that at least part of the direct effect of vitamin D3 metabolites on cell membranes may be related to changes in PA2 activity. The regulation is related to the stage of differentiation of the target cell population. Changes in fatty acid acylation and reacylation may be one mode of vitamin D3 action in cartilage.