The interactions of ceramides with phosphatidate and lysophosphatidate in the regulation of signal transduction in rat fibroblasts were examined. C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) at 10 microM inhibited the stimulation of DNA synthesis that was produced by 50-100 microM phosphatidate, or lysophosphatidate, or by exogenous phospholipase D. Sphingosine (10 microM) had the opposite effect to the ceramides on DNA synthesis. C2- or C6-ceramides failed to inhibit the stimulation of DNA synthesis by insulin or serum. The ceramides did not modify the actions of phosphatidate, or lysophosphatidate, in decreasing the forskolin-induced increase in cAMP. C2- and C6-ceramides inhibited the stimulation of phospholipase D activity by: (a) phosphatidate, lysophosphatidate, phorbol ester, thrombin, or serum in intact fibroblasts and (b) phorbol ester or guanosine 5'-3-O-(thio)triphosphate in permeabilized fibroblasts. The ceramides can therefore modify cell signaling via phospholipase D, but this effect alone could not explain the decreased DNA synthesis. Incubation of fibroblasts with C2- or C6-ceramides or sphingomyelinase inhibited the interaction of exogenous phosphatidate or lysophosphatidate with the fibroblasts by 42 and 53%, respectively. Furthermore, a greater proportion of the phosphatidate, or lysophosphatidate, that was associated with the fibroblasts was metabolized further when the cells were pretreated with ceramides or sphingomyelinase. This effect was accompanied by an increased activity of N-ethylmaleimide-insensitive phosphatidate phosphohydrolase. Ceramides may therefore produce part of their growth inhibitory effects by blocking some of the signal transducing effects of phosphatidate and lysophosphatidate.