Two distinct subsets of human Ia molecules, called NG1 and NG2, present in all individuals irrespective of their HLA-DR phenotype, which were previously defined by their reactivity with two monoclonal hybridoma antibodies, D1--12 and D4--22, were analyzed by two-dimensional peptide mapping techniques. Results show that, in the Ia molecular pool from a single individual, small beta subunits of the NG1 and NG2 subsets display significant differences from each other. In addition, beta subunits of the same subset from two different allotype Ia molecular pools are also different from each other, thus indicating that NG1 and NG2 subsets carry polymorphic specificities. Moreover, large alpha chains of NG1 and NG2 subsets are different from each other; however, no significant differences are observed in alpha chains of the same subset when different allotype Ia pools are analyzed. The possible genetic implications of these findings are discussed.