We designed studies to characterize metabolic aspects of the protective effects of glycine and glutathione against hypoxic proximal tubule cell injury b clarifying the relationship between protection and preservation of tubule cell adenosine triphosphate (ATP) and glutathione levels. The tubule preparation was glutatione depleted as isolated although some recovery occurred during incubation at 37 degrees C, and this recovery was enhanced by treatment with glutatione precursors. Increasing the duration of hypoxia from 30 minutes to 60 minutes produced increasingly extensive lethal tubule cell injury that was almost completely prevented, even at the 60-minute duration, by inclusion of either 2 mmol/L glutathione or 2 mmol/L glycine in the tubule incubation medium. Cell ATP levels decreased to the same extent and at the same rate in protected and unprotected hypoxic tubules. Glycine- and glutathione-protected tubules maintained higher cell glutathione levels than unprotected tubules at all durations of hypoxia studied. However, completely eliminating this increment of glutathione with either the gamma-glutamylcysteine synthetase inhibitor, buthionine sulfoximine, or the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, did not prevent protection. The data indicate that the striking protection against hypoxic injury to the isolated tubules provided by treatment with glycine or glutathione is independent of preservation of tubule cell ATP and glutathione levels, to the extent that difference of these levels can be discriminated in intact cells with present methods.