This report describes for the first time the identification of four forms of biliverdin reductase including two biliverdin-IX beta reductases and two biliverdin-IX alpha reductases, designated isozymes I and II and isozymes III and IV, respectively, in human liver cytosolic fractions. The four forms of biliverdin reductase were purified to homogeneity. There was a 7,800-15,000-fold increase in specific activity when compared with the crude preparation, and the recovery was 8-26%. The purified enzymes were monomers with a molecular weight of about 21,000 (isozymes I and II) and 34,000 (isozymes III and IV). The enzymes were strictly specific for biliverdin, and no other oxidoreductase activities were detected in the purified preparations. The purified enzymes used NADPH and NADH as electron donors for the reduction of biliverdin. The apparent Km values of isozymes I, II, III, and IV for NADPH were 35.9, 13.1, 10.9, and 34.1 microM, respectively, whereas those for NADH were 5.6, 8.2, 7.9, and 23.4 mM, respectively. It was assumed that NADPH rather than NADH was the physiological electron donor in the intracellular reduction of biliverdin. The apparent Km value of isozymes I and II for biliverdin-IX beta in the NADPH system was 0.3 microM whereas those of isozymes III and IV for biliverdin-IX alpha were 1.0 and 0.8 microM, respectively. Isozymes I and II used biliverdin-IX beta, -IX gamma, and -IX delta as substrates but not biliverdin-IX alpha, and isozymes III and IV preferred biliverdin-IX alpha as the most effective substrate among the four biliverdin isomers. The NADPH-dependent enzyme activities were inhibited by substrate concentrations in excess of 3-4 microM. The NADPH-dependent enzyme activities, especially isozymes III and IV, were sensitive to SH reagents including iodoacetamide, p-chloromercuribenzoic acid, and N-ethylmaleimide. The optimum pH of the reaction with NADPH for isozymes I and II was 8.2 whereas that for isozymes III and IV was 7.4. The proportion of the total activity of isozymes I and II to that of isozymes III and IV was considerably higher in the fetal than in the adult liver.