Gametic equality is thought to exist, despite haploid gene action in mammalian spermiogenesis, because of product sharing via the intercellular bridges of conjoined spermatids. However, mice carrying different t-alleles have been known to produce functionally different sperm, leading to transmission ratio distortion (TRD), whose mechanism is unknown. The reduced Spam1 mRNA levels, previously shown to be associated with TRD and reduced fertility in mice carrying the Rb(6.16) or the Rb(6.15) Robertsonian translocation, are reflected in the levels of its encoded membrane protein (Spam1) and its accompanying insoluble hyaluronidase activity. Studies of the temporal expression pattern of Spam1 reveal that it is haploid expressed, with both the RNA and protein first appearing on Day 21.5. RNA fluorescence in situ hybridization and immunocytochemistry show both the mRNA and the protein to be compartmentalized. Compartmentalization of the mRNA along with its immediate translation and insertion of the protein in the plasma membrane suggests the nonsharing of Spam1 transcripts among spermatids, resulting in functionally different sperm in males with different Spam1 alleles. Evidence for biochemically different sperm in these heterozygous males was revealed by flow cytometry and confocal microscopy. Our findings support the notion that the Spam1 antigen is not shared, and we may have uncovered a mechanism for TRD.