Oligodendroglia were isolated from calf central nervous system (CNS) white matter by trypsinization in phosphate buffered saline and separation by centrifugation through Percoll. Using antisera to phenotypic markers and double labelling experiments we were able to identify essentially all cells in the cultures. The cells obtained were: (1) viable; (2) had intact plasma membranes and well preserved organelles, ribosomes and mitochondria; and (3) were greater than or equal to 95% oligodendroglia 16-20 h after isolation as determined by ability to bind antigalactocerebroside antibodies (anti-GalC). Oligodendroglia could be cultured for several weeks to months. Oligodendroglia established and maintained processes which bound anti-GalC. Myelin basic protein could be demonstrated in the cytoplasm of 40-60% of oligodendroglia cell bodies but not in the processes.