Various vasoactive agents (e.g., thrombin and bradykinin) and serum stimulate arachidonate production and thus prostaglandin biosynthesis in cultured fibroblasts. Treatment of 3T3 cells with the anti-inflammatory steroid, dexamethasone, inhibits this stimulation but has no inhibitory effect on the basal activity of phospholipase (or on prostaglandin content) in resting, confluent fibroblasts. In intact cells, the proportion of released arachidonic acid converted into prostaglandins is increased by steroid treatment, in quiescent, dense cells and in serum-treated cells, the total incorporation into prostaglandins is increased. Furthermore, the cyclo-oxygenase activity of homogenates from steroid-treated cells is increased very substantially. Thus, although steroids may affect phospholipase (EC 3.1.1.1.4) activities it is possible that these effects may be secondary to a more important stimulatory effect on cyclo-oxygenase activity which leads to selective alterations in prostaglandin biosynthesis. The steroid-induced increase in cyclo-oxygenase activity is not observed in a transformed variant of the same cell line. Fatty acid lipoxygenase (EC 1.13.11.12) activity exists in the particulate rather than the cytosolic fraction of 3T3 cells.