In this study, we have examined the cellular and biochemical activities of the ceramide analog (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP). Addition of 5 microM D-e-MAPP to HL-60 human promyelocytic leukemia cells resulted in a concentration- and time-dependent growth suppression accompanied by an arrest in the G0/G1 phase of the cell cycle; thus mimicking the action of exogenous ceramides. Its enantiomer L-e-MAPP was without effect. Two lines of evidence suggested that D-e-MAPP may not function as a direct analog of ceramide. First, D-e-MAPP possesses a stereochemical configuration opposite to that of D-erythro-ceramide. Second, D-e-MAPP failed to activate ceramide-activated protein phosphatase in vitro. Therefore, we examined if D-e-MAPP functioned indirectly by modulating endogenous ceramide levels. The addition of D-e-MAPP to cells, but not L-e-MAPP, caused a time- and concentration-dependent elevation in endogenous ceramide levels reaching greater than 3-fold over baseline following 24 h of treatment. Both D-e-MAPP and L-e-MAPP underwent similar uptake by HL-60 cells. D-e-MAPP was poorly metabolized, and remained intact in cells, whereas L-e-MAPP underwent a time- and concentration-dependent metabolism; primarily through N-deacylation. In vitro, L-e-MAPP was metabolized by alkaline ceremidase to an extent similar to that seen with C16-ceramide. D-e-MAPP was not metabolized. Instead, D-e-MAPP inhibited alkaline ceramidase activity in vitro with an IC50 of 1-5 microM. D-e-MAPP did not modulate the activity of other ceramide metabolizing enzymes in vitro or in cells, and it was a poor inhibitor of acid ceramidase (IC50>500 microM). Finally, D-e-MAPP inhibited the metabolism of L-e-MAPP in cells. These studies demonstrate that D-e-MAPP functions as an inhibitor of alkaline ceramidase in vitro and in cells resulting in elevation in endogenous levels of ceramide with the consequent biologic effects of growth suppression and cell cycle arrest. These studies point to an important role for ceramidases in the regulation of endogenous levels of ceramide.