The difficulty in direct detection of oxygen-derived free radicals (OFR) in the intact kidney has left uncertain the role of OFR in renal hypoperfusion injury. Salicylate hydroxylation was used as a sensitive method of estimating the extent of production of highly reactive hydroxyl radicals in renal ischaemia-reperfusion injury in the intact rat kidney perfused with recirculating cell-free medium. The reaction products were detected and quantified by HPLC with electrochemical detection. Hydroxyl radicals were detected as 2,5-dihydroxybenzoic acid (2,5-DHBA). Ischaemia for 15 min followed by reperfusion for 15 min caused more than a twofold increase in 2,5-DHBA concentration (to 2279 +/- 225 pg/g tissue weight) compared to controls (933 +/- 103, P < 0.001). Addition of 15 mM dimethylthiourea (DMTU) before induction of ischaemia prevented this increase. Induction of hypoxia for 15 min with continued perfusion (as a model of low-flow ischaemia) had no significant effect on hydroxyl radical formation. We conclude that significant quantities of hydroxyl radicals form in the absence of circulating leucocytes during reperfusion following ischaemia, but not during hypoxia in the perfused rat kidney.