The H2-M region is the most distal part of the mouse major histocompatibility complex (Mhc) and is likely to include the distal breakpoint of the fourth t-Inversion, In(17)4d. The conserved synteny breakpoint between mouse and human is located in the H2-M region between D17Leh89, a putative olfactory receptor gene, and Pgk2 (phosphoglycerate kinase 2). To analyze the H2-M region, we screened a mouse bacterial artificial chromosome (BAC) library, using the D17Mit64, D17Tu49, D17Leh89, D17Leh467, and Pgk2 markers. Thirty-eight BAC clones were obtained and mapped in five clusters, and 25 sequence-tagged site (STS) markers were newly developed. The regions surrounding D17Tu49 and D17Leh467 are abundant in L1 repeat sequences and may, therefore, be candidates for the breakpoints of conserved synteny and t-inversion. D17Leh89 was linked to D17Mit64 by two contiguous BAC clones. The Aeg1 (acidic epididymal glycoprotein 1) and Aeg2 genes were mapped close to Pgk2, on the same BAC clones. The genetic length between D17Leh89-D17Mit64 and Pgk2-Aeg can be estimated as 0.5–0.7 centiMorgan (cM), and the most distal class I gene, H2-M2, can be placed 0.3–1.0 cM proximal to the t-inversion breakpoint. A recombinational hotspot is suggested to be located between Aeg and Tpx1 in an interspecific cross of (C57BL/6J × Mus spretus).