Previous studies have shown that incubation of rat red blood cells in vitro with phenylhydroxylamine (50-300 microM) induces rapid splenic sequestration of the red cells on reintroduction to isologous rats. EPR and the spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), were utilized to determine if free radical species could be identified under these experimental conditions. Hemolytic concentrations of phenylhydroxylamine, in the presence of DMPO and 5-20% lysed or intact rat erythrocyte suspensions, gave rise to a four-line (1:2:2:1) EPR spectrum. No signal was obtained if phenylhydroxylamine, DMPO, or red cells was omitted. Comparison of the phenylhydroxylamine-induced signal with authentic hydroxyl radical- and GSH thiyl radical-DMPO standard adduct signals identified the phenylhydroxylamine-induced species as a GSH thiyl free radical. Removal of GSH from a red cell lysate abolished the GSH thiyl radical signal without the appearance of any other signal, while addition of exogenous GSH resulted in its return. When erythrocytes were exposed to concentrations of phenylhydroxylamine > or = 200 microM, a time-dependent transition of the GSH thiyl radical signal to a hemoglobin thiyl radical signal was observed. The data are consistent with the postulate that thiyl radical species, generated from the interaction of phenylhydroxylamine and oxyhemoglobin, play a key role in the development of hemolytic injury to the rat red cell.