To investigate the effect of the ceramide moiety of GM1 ganglioside on its association with detergent resistant membrane domains (DRMs) in human leukemia HL-60 cells, [(3)H] labeled GM1 molecular species (GM1s) with ceramides consisting of C18 sphingosine acetylated or acylated with C(8), C(12), C(14), C(16), C(18), C(22), C(24), C(18:1), C(22:1), or C(24:1) fatty acids (FAs), or C20 sphingosine acetylated or acylated with C(8) or C(18) FA were prepared and added to culture media. GM1s uptake by HL-60 cells was affected by the structure of their ceramides. Resistance to removal with trypsin and the stoichiometry of [(125)I] cholera toxin (CT) binding indicated that the added GM1s were incorporated into the membranes of the cells used for the isolation of DRMs in a manner resembling endogenous gangliosides. The ceramide moieties of the GM1s determined their occurrence in DRMs and the dependence of their recovery in this membrane fraction on the amount of Triton X-100 (TX) used for extraction as well as on cholesterol depletion. The GM1s with sphingosine acylated with C(14), C(16), C(18) C(22), or C(24) FAs were similarly abundant in DRMs. GM1s acylated with C(18:1), C(22:1), or C(24:1) were less abundant than those acylated with saturated FA of the same length. GM1s acetylated or acylated with C(8) FA were detected in DRMs in the lowest proportion. Depletion of 73% of cell cholesterol with methyl-beta-cyclodextrin significantly affected the recovery in DRMs of GM1s acetylated or acylated with C(8) or unsaturated FAs but not of GM1 acylated with C(18), C(22), or C(24) FAs. After cross-linking with CT B subunit, all GM1s were recovered in DRMs in a similarly high proportion irrespective of their ceramide structure or cholesterol depletion. DRMs prepared with low TX concentration at the TX/cell protein ratio of 0.3:1 were separated by multistep sucrose density gradient centrifugation into two fractions. The GM1s with sphingosine acetylated or acylated with C(18) or C(18:1) FAs occurred in these fractions in different proportions.