Previous studies have shown that phospholipase A2 activity in rat costochondral chondrocyte cultures is differentially regulated by 1,25-(OH)2D3 and 24,25-(OH)2D3. 1,25-(OH)2D3 stimulates enzyme activity in growth zone chondrocytes but has no effect on the resting zone chondrocyte enzyme activity. 24,25-(OH)2D3 inhibits the resting zone enzyme but has no effect on the growth zone chondrocyte phospholipase A2. This study examined whether the metabolites affect arachidonic acid turnover in their target cell populations. Incorporation and release of [14C]arachidonate was measured at various times following addition of hormone to the cultures. Acylation and reacylation were measured independently by incubating half of the [14C]arachidonate-labeled cultures with p-chloromercuribenzoate. The results demonstrated that the distribution of [14C]arachidonate in membrane phospholipids differed between growth zone and resting zone chondrocytes and between the plasma membranes and matrix vesicles isolated from the growth zone chondrocyte cultures. Plasma membrane phospholipids were more susceptible to the release of [14C]arachidonic acid by exogenous phospholipases than were matrix vesicle phospholipids. The effect of 1,25-(OH)2D3 on growth zone chondrocytes was observed within 5 min. Incorporation was greatest after 60 min; release was greatest after 30 min. 24,25-(OH)2D3 stimulated consistently elevated incorporation throughout the incubation period, peaking at 15 min. Peak release was at 60 min. The results confirm that resting zone chondrocytes and growth zone chondrocytes retain a differential phenotype in culture and demonstrate that matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25-(OH)2D3 and 24,25-(OH)2D3 appear to stimulate arachidonic acid turnover in their target cells by different mechanisms. Changes in fatty acid acylation and reacylation may be one mode of vitamin D-3 action in cartilage.