In previous work certain hydroxylated and peroxylated derivatives of uroporphyrin (URO) have been isolated from the urine of patients suffering from porphyria. We have now investigated the mechanism of production of these oxygenated derivatives of URO, using both enzymic and chemical model systems and also the effect of exposure to light during reoxidation of uroporphyrinogen (URO'gen). When URO'gen was incubated with haemolysates, peaks with the same retention times as peroxyacetic acid URO, meso-hydroxy URO and /3- hydroxypropionic acid URO were all detected. The first of these was formed in sufficient amounts to allow its characterization by mass spectrometry. Under these conditions, peroxyacetic acid derivatives of heptacarboxylate and pentacarboxylate porphyrins could also be produced from the corresponding porphyrinogens, but no peroxylated product could be obtained from coproporphyrinogen (COPRO'gen, where no acetic acid side chains are present) or from the fully oxidized URO. Similar results were obtained on re-oxidation of URO'gen in the xanthine oxidasexanthine system and in the presence of hydrogen peroxidme-EDTA (ethylenediamine-tetraacetic acid) and here again no peroxylated product could be detected from either COPRO'gen or URO. Finally, formation of peroxyacetic acid URO could be demonstrated during photo-oxidation of URO'gen and this was followed by light-induced loss of both URO and its peroxylated derivative. It is concluded that the oxygenated derivatives arise from the action of reactive oxygen species on the porphyrinogens (rather than the porphyrins), with one of the acetic acid side chain serving as the preferential (or exclusive target) for peroxylation.