Previous studies have shown that treatment of cultured rabbit coronary microvessel endothelial (RCME) cells with dexamethasone results in a dose-, time-, and glucocorticoid-dependent inhibition of prostaglandin release. In the present study, the effects of dexamethasone on RCME membrane lipid composition and release of arachidonic acid were examined. This study demonstrated that dexamethasone treatment did not significantly alter the relative distribution of membrane phospholipids but did result in changes of fatty acid composition. There was an increase in saturated and monounsaturated fatty acids and a decrease in polyunsaturated fatty acids. Dexamethasone treatment did not reduce A23187-stimulated arachidonic acid release, despite inhibiting prostaglandin release by 50%. Studies with radiolabeled arachidonic acid suggest that dexamethasone may exert some actions on membrane remodeling, an effect that will require further investigation. Our data strongly suggest that the inhibitory actions of glucocorticoids on prostaglandin release in cultured RCME cells are not the result of a generalized inhibition of arachidonic acid release, and alternate mechanisms must therefore be considered.