The production of PGE 2 by chondrocytes and its regulation by vitamin D metabolites was examined in this study as a function of cell maturation. Costochondral chondrocytes, derived from the resting zone and growth zone cartilage, were grown in culture to fourth passage. At confluence, they were exposed to 10 −8−10 −11M 1,25-(OH) 2D 36 or to 10 −7−10 −10M 24,25-(OH) 2D 3 for either five minutes or 3,6,12, or 24 hours. Indomethacin (10 −7M) was added to one-half of the cultures to block the production of PGE 2. The amount of PGE 2 released into the media was determined by radioimmunoassay. Both growth zone and resting zone cells produced PGE 2 in a time-dependent manner; PGE 2 concentration was greater in the resting zone cell cultures. 1,25-(OH) 2D 3 stimulated PGE 2 production by growth zone cells in a dose-dependent manner, significant at 10 −8−10 −10M. This effect was observed at 3 hours and remained elevated during the 24 hours of culture. 1,25-(OH) 2D 3 had no effect on PGE 2 production by resting zone cells. However, 24,25-(OH) 2D 3 (10 −7−10 −8M) inhibited PGE 2 production from 3–24 hours. No effect was noted when 24,25-(OH) 2D 3 was added to growth zone cells. Indomethacin reduced PGE 2 production to baseline values in all groups examined. The results indicate that chondrocytes in culture produce PGE 2. Production is regulated by vitamin D 3 metabolites and is cell maturation-dependent. The correlation between hormone-dependent PGE 2 production and phospholipase A 2 activity suggests that the effects of 1,25-(OH) 2D 3 and 24,25-(OH) 2D 3 on chondrocytes may be mediated, in part, by changes in phospholipase A 2 activity, arachidonic acid release, and PGE 2 production. PGE 2 may then act as a second messenger in a paracrine or autocrine manner.