Ceramide is produced in HL 60 cells in response to 1 alpha,25-dihydroxy vitamin D3 (vitamin D3) (Okazaki et al. (1989) J. Biol. Chem. 264, 19076-19080). HL 60 cell differentiation can be mimicked by cell permeable ceramides (Okazaki et al. (1990) J. Biol. Chem. 265, 15823-15831). Vitamin D3 like other thyroid-steroid hormones binds to a member of the steroid hormone family. We sought to investigate whether agonists other than vitamin D3 which exert their effects through members of the steroid receptor family affect cellular ceramide levels. Treatment of GH4C1 cells with 10 microM all-trans retinoic acid for 8 h caused a 230% increase in cellular ceramide content. This concentration of retinoic acid also inhibited cell proliferation as measured by [3H]thymidine incorporation. Under these conditions, a 35% decrease in sn-1,2-diacylglycerol mass occurred, but no change in sphingomyelin, sphingosine or phosphatidylcholine mass was observed. To determine the mechanism of increased ceramide production by 10 microM retinoic acid, cells were labeled with [3H]palmitic acid. After a 2 h period, a 4-fold increase in the incorporation of palmitate into ceramide was observed. Hydrolysis of the labeled ceramide to sphingosine and free fatty acid demonstrated that only 6% of the label was recovered in the sphingosine backbone of cells treated with retinoic acid, whereas 20% of the label was recovered in the sphingosine backbone of cells treated with vehicle alone. The data are consistent with retinoic acid causing an increase in cellular ceramide levels in GH4C1 cells through an increase in sphingosine N-acylation.