Growth of Chinese hamster ovary and LM cells is inhibited by relatively low concentrations of sphingosine in the culture media. This effect is diminished by an order of magnitude by conversion of this positively charged long-chain amino alcohol to a number of N-acetylated analogues, such as N-acetylsphingosine, N-acetylsphingosine phosphate, and N-acetylsphingosine phosphorylcholine. Synthesis of sphinganine and its incorporation into ceramide, sphingomyelin, and glycosphingolipids (GSL) was monitored using a short pulse of [14C]serine together with a long pulse of [3H]-galactose. Compared to unsupplemented cultures, growth with 15-30 microM N-acetylsphingosine suppressed incorporation of 14C radioactivity into ceramide, sphingomyelin, and GSL by 75-95% without accumulation of labeled sphinganine and without any appreciable change in membrane phospholipid or total GSL content. Furthermore, when cells were cultivated with 15 microM [4,5-3H]N-acetylsphinganine to monitor its utilization for sphingolipid synthesis, considerable loss of radiolabel occurred due to desaturation of sphinganine to sphingosine. Nevertheless, most of the residual label was found in the long-chain base and not the acyl group of sphingomyelin, indicating that the exogenously supplied base was utilized intact for complex lipid synthesis. Radiolabel was also found in ceramide and glycosphingolipid fractions. Thus, established cell lines whose growth is very sensitive to long-chain amino alcohols can be cultivated with sphinganine (sphingosine) analogues at concentrations which suppress endogenous sphinganine production but support continued synthesis of complex sphingolipids.