Prior studies have shown that 24,25-(OH) 2 D 3 and 1,25-(OH) 2 D 3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH) 2 D 3 affecting primarily growth zone (GC) cells and 24,25-(OH) 2 D 3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH) 2 D 3 has been shown to increase phospholipase A 2 activity in GC, while 24,25-(OH) 2 D 3 has been shown to decrease phospholipase A 2 activity in RC. Stimulation of phospholipase A 2 in GC caused an increase in PKC, whereas inhibition of phospholipase A 2 activity in RC cultures increased both basal and 24,25-(OH) 2 D 3 -induced PKC activity, suggesting that phospholipase A 2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A 2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A 2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A 2 inhibitors decreased both basal and 1,25-(OH) 2 D 3 -induced PKC activity in GC. When phospholipase A 2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH) 2 D 3 -induced PKC activity in RC and increased 1,25-(OH) 2 D 3 -induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A 2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A 2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A 2 . These effects are cell maturation-and time-dependent and metabolite-specific.