The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [3H]arginine to [3H]citrulline. Cytosolic Ca2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1-40 microM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.