It has been hypothesized that complex sphingolipids may serve as another "lipid second messenger" system via their hydrolysis to free sphingosine, which inhibits protein kinase C and affects multiple cellular functions. To investigate sphingolipid turnover, Chinese hamster ovary cells were pulse labelled with [14C]serine and the [14C]sphingosine in cellular sphingolipids was determined over time. Much of the radiolabelled sphingosine was initially seen in ceramides and was incorporated into sphingomyelin during the 5-hour chase. A major portion of the radiolabel that was initially seen in other sphingolipids disappeared over time. Overall, about half of the total long-chain bases made during this pulse were degraded within 2 to 5 h, depending on the method of analysis. Hence, a substantial portion of the sphingosine synthesized de novo by these cells is turned over fairly quickly. Since the doubling time of these cells is 12 h, this rapid turnover may reflect the remodelling of the cell surface, or the utilization of the free sphingosine derived from sphingolipid turnover, as part of the control of cell growth and division.