A method is described for the isolation of fractions rich in prolactin (PRL) granules from adenohypophyses of female rat. The method consists of three steps: (a) differential centrifugation; (b) incubation of a post nuclear pellet, enriched in PRL granules, in a medium containing puromycin at high ionic strength in order to decrease the density of the contaminating rough microsomes, by detaching the membranebound ribosomes and to eliminate most of the other contaminants (growth hormone granules and mitochondria), by changing their sedimentation properties; (c) isopycnic centrifugation in a discontinuous sucrose density gradient. The PRL granules isolated by this method are relatively free of contaminants, as indicated by ultrastructural and biochemical criteria. (Endocrinology 92: 349, 1973)