Methemoglobinemia is induced by aminoazo dyes in rats of the Sprague-Dawley strain. 4-Anunoazobenzene (AB) induces about 66.6% of methemoglobin (MHb) by i.p. injection of a dose of 3.23 × 10-4 moles/kg, while in the same dose, N-methyl- and N, N-dimethyl-4-aminoazobenzene cause 51.7 and 22.3% maximum levels of MHb respectively. The MHb-forming activity of the metabolites of AB in rats and in isolated rat erythrocytes has been investigated. N-OH-AB seems to be the most active metabolite of AB in causing methemoglobinemia in rats and in inducing MHb in isolated erythrocytes. The reductive cleavage products of AB (aniline, p-aminophenol and p-phenylenediamine) are not the main MHb formers in the early stage, but they may contribute partially to the MHb concentration in the later period of methemoglobinemia induced by AB in vivo. The ring-hydroxylated metabolites of AB, such as 4′-OH-AB and 3-OH-AB, also have MHb-forming activity, but are not so active as N-OH-AB. N-benzoyloxy-N-methyl-4-aminoazobenzene and N,N-dimethyl-4-aminoazobenzene-N-oxide are quite active in inducing MHb in the isolated rat erythrocytes.