Ascorbic acid and glutathione deficiencies during the isolation of tissue cells could compromise metabolic functions involving their participation. This possibility was assessed by supplementing the medium used in the isolation of rabbit pulmonary type II cells with varying concentrations and combinations of ascorbic acid, glutathione, and glutathione's constituent amino acids, glutamine, cysteine, and glycine. Isolated type II cells were monitored for changes in their biosynthetic capabilities by examination of 3H-choline and 3H-leucine incorporation into phospholipids and proteins. In the presence of glutathione's constituent amino acids and low concentrations of ascorbic acid (0.056 and 0.56 mM) incorporation of radiolabeled choline and leucine by isolated type II cells was decreased by 50% compared with unsupplemented controls. Morphologic examination revealed that type II cells isolated in the presence of glutathione's constituent amino acids had reduced surface membrane blebbing and dilation of endoplasmic reticulum cisternae compared with unsupplemented controls. Intracellular glutathione levels in these cells was also increased by 100% compared with those isolated without the constituent amino acids. Type II cells isolated in the presence of ascorbic acid and the constituent amino acids attached to culture dishes with a 5-fold greater efficiency compared with unsupplemented controls. The supplementation of type II cell's isolation media with ascorbic acid and glutathione's constituent amino acids produces several beneficial affects possibly through reducing intracellular oxidant damage, although other mechanisms of action cannot be ruled out.