In mice bearing the Rb(6.16) or Rb(6.15) Robertsonian translocation (Rb), sperm dysfunction associated with the Rbs has been shown to lead to transmission ratio distortions (TRDs) in heterozygotes. The severity of the TRDs is directly related to the severity in the alteration of expression of the gene for the Sperm Adhesion Molecule 1 (Spam1), which maps to proximal mouse Chromosome 6 (Chr 6) near the translocation junction and encodes a sperm antigen with hyaluronidase activity. Here we demonstrate that there is a significantly reduced fertility in the Rb homozygotes (P < 0.001), based on litter size; and that with the Sperm Select Penetration assay Rb-bearing sperm have significantly decreased (P < 0.02-0.001) rates of penetration of hyaluronic acid. Catalytic kinetics studies indicate that reduced Spam1 (PH-20) hyaluronidase activity in the Rb(6.15) mice results from a qualitative defect, while for Rb(6.16) with the greater TRD both a qualitative and a quantitative deficiency (confirmed by Western analysis) of Spam1 exist. Six point mutations were shown to be clustered in the Spam1 hyaluronic acid-binding domain in Rb(6.15). For Rb(6.16) which has a gross genomic alteration at the Spam1 locus, 11 point mutations are scattered in the 5' and 3' UTRs and the coding region, where one leads to the replacement of a conserved residue. Entrapment of spontaneous Spam1 mutations, owing to recombination suppression near the Rb junctions, is proposed as the major underlying defect of the sperm dysfunction.