The possible relationship between metabolism and biological effects of sphingosine was investigated in Neuro2a cells. [C3‐³H]‐sphingosine, administered at different doses (80 pmol‐80 nmol/mg cell protein). Amounts up to hundredfold were rapidly taken up and metabolized, the intracellular content of sphingosine being processed within 2 h. At low doses, [³H]‐sphingosine represented a minor portion of the cellular radiolabel, and N‐acylated metabolites, particularly ceramide, prevailed over degradation products. Neuro2a cell differentiation took place in conjunction with ceramide increase. At increasing exogenous sphingosine/cell ratio, the acylation process became saturated while sphingosine degradation increased proportionally. From this point on [³H]‐sphingosine accumulated and cell toxicity occurred. In conclusion, in Neuro2a cells the biological effects exerted by exogenous sphingosine are strictly connected to the exogenous sphingosine/cell ratio and to the capacity of the cell to metabolize sphingosine.