Abstract A fraction rich in membranes of the Golgi apparatus was isolated from rat brain by discontinuous density gradient centrifugation. The fraction sedimented at the characteristic Golgi density of 1.11–1.15 (g/cm 3, 5°C) and had specific activities of Golgi-marker enzymes ( N-acetyllactosaminyl synthetase, glycoprotein (Fetuin) galactosyltransferase, thiamine pyrophosphatase), 6–7 times over those of th original homogenates. The recovery of the enzyme activities in this fraction ranged from 17 to 31%. The incorporation [ 3H]fucose into glycoproteins was 3-fold higher than in homogenate. Recovery and relative specific activities of marker enzymes for other subcellular organelles were low. Electron microscopic analysis of the fraction revealed the presence of Golgi structures, namely, large sacs or plates with attached tubules and “blebbing” of the tubules into the vesicles.