Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 x 10(6) cells per rat, of which 50 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 x 10(6) cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 +/- 5 per cent) excluded the vital dye, trypan blue. The cells consumed O2 at the rate of 76 +/- 12 nmole per 10(6) cells per hour and released only 5.7 +/- 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after 1 hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their O2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.