HLA class II alpha and beta chains form receptors for antigen presentation to CD4(+) T cells. Numerous pairings of class II alpha and beta subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched alphabeta heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study we investigated assembly of isotype-mixed HLA class II alpha and beta heterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed alpha and beta subunits was dependent on the groove binding section of the invariant chain (Ii). By mutation of polymorphic DPbeta sequences, we identified two motifs, Lys-69 and GGPM-(84-87), that are engaged in Ii-dependent assembly of DPbeta with DRalpha. We identified five members of a family of DPbeta chains containing Lys-69 and GGPM 84-87, which assemble with DRalpha. The Lys/GGPM motif is present in the DPbeta sequence of the Neanderthal genome, and this ancient sequence is related to the human allele DPB1*0401. By site-directed mutagenesis, we inspected Neanderthal amino acid residues that differ from the DPB1*0401 allele and aimed to determine whether matched heterodimers are formed by assembly of DPbeta mutants with DRalpha. Because the *0401 allele is rare in the sub-Saharan population but frequent in the European population, it may have arisen in modern humans by admixture with Neanderthals in Europe.