Recognition of foreign antigen by cytotoxic T lymphocytes (CTL) is restricted by class I major histocompatibility complex (MHC) products. Class I heavy chains (relative molecular mass (Mr) 45,000-48,000) are reversibly and noncovalently associated with beta 2-microglobulin (beta 2M, Mr = 12,000). Cells expressing human or murine class I heavy chains can exchange their native beta 2M for exogenously added free beta 2M, which is present in serum. Two allelic forms of beta 2M exist among the common laboratory mouse strains, beta 2M-A and beta 2M-B, which are represented in BALB and C57BL mice, respectively. The two forms differ at a single amino acid at position 85, the gene (beta 2m) is located on chromosome 2 linked to a minor histocompatibility (H) region, H-3. It has been proposed that one of the H-3 loci is identical with beta 2m, and that CTL raised across certain H-3 incompatibilities are actually specific for beta 2M. Here we describe CTL raised in such a combination which recognize endogenous as well as exogenous beta 2M-B in the context of H-2Kb. This represents a unique case of CTL recognition, as CTL usually recognize antigens inserted into the membrane, and it is the first molecular identification of the product of a minor H locus.