cDNAs corresponding to three human runt domain containing genes, AML1, AML2, and AML3, were isolated and characterized. In addition to homology in the highly conserved runt domain, extensive sequence similarities were also observed in other parts of the proteins. All three carried an identical, putative ATP binding site -GRSGRGKS-, and their C-terminal halves were particularly rich in proline and serine residues. While AML1 cDNAs were cloned by others, AML2 represents a new member, not previously described, of the runt domain gene family, and AML3 was identified as the human homologue of mouse PEB-P2 alpha A. The chromosomal location of AML1 to chromosome 21q22 was confirmed, while AML2 and AML3 were mapped to chromosome regions 1p36 and 6p21, respectively. Analysis of AML1 and AML2 expression in hematopoietic cell lines revealed a distinct pattern of expression.