Isoelectric focusing in the ultranarrow immobilized (7.1-7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different II alpha-globin chains were separated from two different I alpha-chains by HPLC. Two alpha-chains (I alpha 1 and II alpha 3), and Hbs with similar mobilities (Hb1 and Hb3), were associated with the AA Hb phenotype: two alpha-chains (I alpha 2 and II alpha 4), and Hbs with different mobilities (Hb2 and Hb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two alpha loci. An allele at the beta locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.