In this study, analogs differing at the C-1 or C-3 position of the sphingosine backbone of sphingomyelin were examined in neutral pH-optimum sphingomyelinase assays. Two analogs modified at the C-1 position, ceramide-1-phosphate and ceramide-1-phosphoethanol-N,N-dimethylamine, could act as modest substrates but showed no ability to inhibit the reaction when egg sphingomyelin was used as the substrate. Four analogs of sphingomyelin differing at the C-3 position were used in which the hydroxyl group was replaced by a hydrogen atom (to give a deoxy-sphingomyelin analog), or with a O-methyl, O-ethyl or O-tetrahydropyranyl group. The deoxy analog showed no ability to compete with substrate of sphingomyelinase nor could it be hydrolyzed by the enzyme, suggesting that the hydroxyl group is a required substituent for the substrate. The 3-O-methyl and 3-O-ethyl-sphingomyelin analogs were inhibitors, with IC50 values of 50 microM and 140 microM, respectively at standard assay conditions. However, when the rat brain acidic pH-optimum sphingomyelinase was used, no inhibition by the 3-O-methyl analog could be detected. The size of the alkyl group on the ether moiety was important, as shown by the inability of 3-O-tetrahydropyranyl-sphingomyelin to compete with substrate of neutral pH-optimum sphingomyelinase.