1a,25-(OH) 2 D 3 mediates its effects on growth zone chondrocytes via rapid membrane-associated events as well as through traditional nuclear receptor mechanisms. The membrane-associated signaling pathways include rapid production of diacylglycerol and activation of protein kinase C (PKC), as well as activation of phospholipase A 2 (PLA 2 ), increased production of arachidonic acid, and increased production of prostaglandins. This study examined the roles of PLA 2 and cyclooxygenase (Cox) in the mechanism of action of 1a,25-(OH) 2 D 3 in these cells to determine whether one or both enzymes catalyze the rate limiting step and whether constitutive or inducible Cox is involved. Cultures were incubated with 1a,25-(OH) 2 D 3 for 9 min to measure PKC or for 24 h to measure physiological responses ([ 3 H]-thymidine incorporation, alkaline phosphatase speci®c activity, [ 35 S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, growth zone chondrocytes expressed mRNAs for both Cox-1 and Cox-2 and neither Cox was modulated by 1a,25-(OH) 2 D 3 . To examine the role of Cox, the cultures were also treated with resveratrol (a speci®c inhibitor of Cox-1), NS-398 (a speci®c inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). The results showed that Cox-1 inhibition reduced the 1a,25-(OH) 2 D 3 -dependent effects on proliferation, differentiation, and matrix production, whereas inhibition of Cox-2 only had an effect on proliferation. The effects of Cox inhibition were not rate limiting, based on experiments in which PLA 2 was activated with melittin or inhibited with quinacrine. However, at least part of the action of 1a,25-(OH) 2 D 3 was regulated by metabolism of arachidonic acid to prostaglandins. This supports the hypothesis that 1a,25-(OH) 2 D 3 exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA 2 as a rate-limiting step. PKC regulation may occur at multiple stages in the signal transduction cascade.