A procedure has been developed for the rapid isolation of relatively pure populations of glia, Müller cells, from mammalian retina and is reported here for the rabbit. The retina, cleaned of vitreous, is dissociated by 4 interacting operations--enzymatic digestion of extracellular matrix by means of hyaluronidase, collagenase and papain, removal of divalent cations, acidification and mild trituration. The resultant admixture consists of receptors, neurons and glia; 10% of the cells are Müller cells. These glial elements can be brought to 95% purity by rapid centrifugation on convex 0-30% Percoll gradients. Of the resultant glia, 80% exclude trypan blue. The Müller cells are enriched in two glial specific enzymes, glutamine synthetase and carbonic anhydrase and they retain a significant fraction of the membrane bound carbonic anhydrase enzyme activity. Light and scanning electron microscopy show the cells to be well preserved and covered extensively with microvilli. In the outer nuclear zone, the cells are plicated and end in bulbs tufted with microvilli. The procedure we describe allows studies of a new preparation of intact, relatively undamaged, adult, isolated mammalian Müller cells to better understand the functioning of glial cells.