The various manipulations involved in the isolation of membrane fragments from culture fluids of murine melanoma cells were examined to discern their effect on membrane fragment structure. Ultracentrifugation and gel chromatography were compared using the presence of marker enzymes and the sensitivity to a non-ionic detergent (Triton X-100). Fractionation of media by gel chromatography resulted in only one major form of membrane particles, while ultracentrifugation, followed by resuspension, produced at least two major populations from the identical material. These results indicate that the optimal procedure for membrane fragment isolation is fractionation by an agarose-containing gel, followed by concentration using PEG 20,000.