Ceramide and ceramide-1-phosphate are sphingolipid analogues of diacylglycerol and phosphatidate, respectively, and they are putative second messengers of agonist-stimulated sphingomyelin metabolism. The interactions of exogenous cell-permeable ceramides and ceramide-1-phosphates in modifying DNA synthesis and signal transduction were investigated in Rat-1 fibroblasts. C2- and C8-Ceramide-1-phosphates (N-acetylsphingosine-1-phosphate and N-octanoylsphingosine-1-phosphate, respectively) at 1-10 microM stimulated DNA synthesis and cell division. This effect was blocked by cell-permeable ceramides. C2-Ceramide stimulated the conversion of exogenous C8-ceramide-1-phosphate to C8-ceramide, with very little production of sphingosine or sphingosine-1-phosphate. This mechanism may be partly responsible for preventing the stimulation of DNA synthesis. Unlike phosphatidate or lyso-phosphatidate, concentrations of C8-ceramide-1-phosphate that stimulated DNA synthesis did not inhibit adenylate cyclase activity, nor did they increase the activities of phospholipase D or mitogen-activated protein kinases (42- and 44 kDa isoforms). Although ceramide-1-phosphate can be considered as an analogue of phosphatidate, the effects of this compound on signal transduction differ considerably from those of phosphatidate. This work demonstrates that short-chain ceramide-1-phosphates can be used as novel external agonists that can stimulate DNA synthesis. This effect can be counteracted by short-chain ceramides.