Four of the eleven independent H-2 haplotypes of inbred mouse strains and approximately 15% of wild mouse chromosomes 17 fail to express the E alpha E beta class II histocompatibility (Ia) Ag. These E- haplotypes are defective in the expression of the E alpha and/or the E beta chain. None of the E beta defects has previously been described at the molecular level. In this study, we report the molecular basis for the defective expression of the E beta gene from the w17 haplotype of the H-2 congenic strain B10.CAS2, derived from wild Mus musculus castaneus. Comparison of the Ew17 beta genomic sequence to those of the functional Eb beta and Ed beta genes reveals a single base insertion in the RNA donor splice site of the first intron. By DNA shuffling, we have corrected the single base mutation, and we show by FACS analysis and 2-D PAGE of immunoprecipitates that the corrected Ew17 beta is expressed in L cells when co-transfected with an Ed alpha gene. Conversely, an Eb beta gene construct containing the mutant RNA splice site from Ew17 beta is not expressed. We conclude that the single base insertion in the first RNA splice donor site is the sole molecular defect in the Ew17 beta gene.