The effect of ceramide on nitric oxide (NO) formation was studied in rat alveolar macrophages (AMs). Rats were infused with ethanol (EtOH) for 3 h, or the EtOH infusion was combined with i.v. injection of endotoxin (ET) 90 min into the infusion. Controls were infused with saline. Alveolar macrophages were obtained by bronchoalveolar lavage and were cultured for 20 h in the presence and absence of ET, interferon-gamma (IFN), C6 ceramide (N-hexanoylsphingosine), and C2 dihydroceramide. Nitric oxide formation was assessed by measurement of nitrites in the medium. C6 ceramide significantly suppressed NO formation in response to in vitro addition of ET, but not IFN. C2 dihydroceramide caused no inhibition. The ceramide effect appears to be not only stimulus specific, but also specific to AMs, since NO formation by Kupffer cells and liver infiltrated neutrophils was not affected. The results suggest involvement of the sphingomyelin cycle in ET-stimulated NO formation in rat AMs.