Axolemmal fractions are isolated by discontinuous gradient centrifugation of purified myelinated axons which have been osmotically shocked to yield axons, myelin and axolemma. The axolemmal fractions are enriched in surface membrane marker enzymes such as Na(+),K(+)-activated ATPase, 5'-nucleotidase and acetylcholinesterase. Mixing experiments with labeled microsomes and mitochondria show that these contaminants are effectively screened out by the preparation procedure. The fractions contain significant amounts of 2',3'-cyclic nucleotide-3'-phosphohydrolase and are enriched in higher molecular weight proteins. These membrane preparations may be useful in analytical studies of mammalian axolemmal composition.