When incubated with a hydroxyl radical (HO.)-generating system (ascorbic acid/Fe(2+)-EDTA/O2/H2O2), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3'-bis(2-aminoethyl)-5-hydroxy-[3,7'-bi-1H-indole]-2,4',5'- 3H-trione (7). The latter slowly cyclizes to 3'-(2-aminoethyl)-1',6',7',8'-tetrahydro-5-hydroxy-spiro[3H-indole-3,9'- [9H]pyrrolo[2,3-f]quinoline]-2,4',5' (1H)-trione (9). A minor amount of T-4,5-D dimerizes to give 7,7'-bi-(5-hydroxytryptamine-4-one) (7,7'-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO.-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO. formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO.-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO. formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO. might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.