Third passage confluent cultures of cartilage cells, initially derived from the growth zone (GC) and resting zone (RC) of rat costochondral cartilage, were incubated with either 10(-11)-10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or 10(-9)-10(-6) M 24,25-(OH)2D3. Plasma membranes and extracellular matrix vesicles were isolated, and specific activities of phospholipase A2 and alkaline phosphatase were determined. The results demonstrate that the response to hormone is both cell and membrane specific. 1,25-(OH)2D3 produces an increase in GC matrix vesicle alkaline phosphatase and phospholipase A2 specific activities at 10(-9) and 10(-8) M, but has no effect on these enzyme activities in RC membranes. RC cultured in 24,25-(OH)2D3 exhibit increased matrix vesicle alkaline phosphatase but decreased phospholipase A2 activities at 10(-7) and 10(-6) M hormone. No effect on the RC plasma membrane enzymes or on GC plasma membrane or matrix vesicle enzymes was observed. The data suggest that changes in membrane fluidity due to phospholipase A2 activity may play a role in regulating alkaline phosphatase activity in response to vitamin D metabolites and that this regulation in GC and RC may proceed by different mechanisms.