Minor histocompatibility (H) loci are significant tissue transplantation barriers but are poorly understood at the genetic and molecular level. We describe the construction of a high-resolution genetic map that positions a class II MHC-restricted minor H antigen locus and orders 12 other genes and genetic markers within the we-un interval of mouse Chromosome (Chr) 2. An intersubspecific backcross between B10.UW/Sn-H-3b and CAST/Ei, an inbred stock of Mus musculus castaneus, was used for this purpose. A total of 1168 backcross mice were generated, and 71 we-un recombinants were identified. Significant compression of the genetic map in males versus females and transmission distortion of CAST-derived we, un, and Aw genes were observed. Monoclonal T cell lines specific for two minor H alloantigens, Hd-1a and Hd-2a, encoded by gene(s) that map to the we-un interval were used to antigen type the backcross mice. The results suggest the Hd-1a and Hd-2a antigens are most likely encoded by a single gene, now referred to as H-3b. The determined gene order is we-0.09 +/- 0.09-Itp-0.62 +/- 0.23-D2Mit77-0.26 +/- 0.15-[Evi-4, Pcna, Prn-p]-0.26 +/- 0.15-Scg-1-0.44 +/- 0.19-[Bmp2a, D2Mit70]-0.09 +/-. 0.09-[D2Mit19, D2Mit46]-1.59 +/- 0.36-D2Mit28-0.97 +/- 0.28-D2Ler1-1.50 +/- 0.35-H-3b-0.26 +/- 0.15-un (% recombination +/- 1 SE). Because the average resolution of the backcross is 0.09 cM, the backcross panel should facilitate the physical mapping and molecular identification of a number of genes in this chromosome region.