We have constructed a new series of hybrid genes among the H-2Kd,-Kk, and -Kb. The site of recombination occurs in the third exon, encoding the alpha 2 domain, and divides this domain into two parts, alpha 2A and alpha 2B. The novel genes differ only in the COOH-terminal half of the alpha 2 domain, i.e., the alpha 2B region. This region, comprising residues 142-182, contains a limited number of amino acid differences between the three alleles. The hybrid genes have been introduced into 1T 22-6 cells (H-2q), and cell surface expression of hybrid antigens was verified. Cells expressing different types of hybrid antigens have been examined for their susceptibility to lysis by cytotoxic T lymphocytes directed either against the H-2Kd antigen or the H-2Kk antigen. Our results show that the alpha 1 and alpha 2A domains of the H-2Kk antigen can constitute target molecules for alloimmune anti-Kk T cells, whereas the alpha 2B region, when exchanged for Kd or Kb sequences, plays only a limited role. In contrast, the alpha 1 and alpha 2A domains of Kd are not sufficient to be recognized by alloimmune anti-Kd T cells. In this instance, the alpha 2B domain seems to play an essential role. This region has undergone several amino acid substitutions involving charged residues.