Supplementation of the diet of CF1 mice with sphingomyelin isolated from milk has been shown to reduce the number of aberrant crypt foci (ACF) and the appearance of colonic adenocarcinoma induced by 1,2-dimethylhydrazine (Schmelz et al., Cancer Res 56, 4936-4941, 1996). The objective of this study was to determine whether chemically synthesized sphingomyelin reduces the appearance of ACF, one of the earliest morphological changes in the development of colonic tumors, and to investigate the specificity of this inhibition for the unsaturated sphingoid base backbone. 1,2-Dimethylhydrazine was administered intraperitoneally to female CF1 mice, then the animals were fed a semipurified AIN 76A diet without supplementation (controls) or supplemented with 0.1% (wt/wt) sphingomyelin isolated from skim milk powder, synthetic N-palmitoylsphingomyelin, or N-palmitoyldihydrosphingomyelin for four weeks. The number of ACF in the sphingomyelin-fed groups was significantly lower than in the control by 54% (p = 0.002), 52% (p = 0.002), and 70% (p < 0.0001) for milk sphingomyelin, synthetic sphingomyelin, and synthetic dihydrosphingomyelin, respectively. Suppression of ACF by the synthetic dihydrosphingomyelin was significantly greater than by synthetic sphingomyelin (p = 0.035). These findings establish that sphingomyelin, and not merely a possible contaminant of the naturally occurring sphingomyelin preparation used previously, suppresses ACF formation. Furthermore, the greater potency of dihydrosphingomyelin reveals that the 4,5-trans double bond of the sphingoid backbone is not required for this suppression.