CABYR is a highly polymorphic, sperm flagellar calcium-binding protein that is tyrosine as well as serine/threonine phosphorylated during capacitation. Six alternative splice variants of human CABYR (I-VI) have previously been identified, involving two coding regions, CR-A and CR-B, separated by an intervening stop codon. It is presently unknown if proteins encoded by the predicted coding region B of CABYR are translated during spermiogenesis, where they localize, or which CABYR isoforms bind calcium. Immunofluorescent and electron microscopic studies using polyclonal antibodies generated to the recombinant c-terminal 198 aa CABYR-B localized the isoforms containing CABYR-B to the ribs and longitudinal columns of the fibrous sheath in the principal piece of the flagellum. Antisera to recombinant CABYR-A and CABYR-B proteins recognized distinct populations of CABYR isoforms encoded by either CR-A alone and/or CR-B as well as a common population of CABYR isoforms. Only the recombinant CABYR-A and not the CABYR-B bound calcium in vitro, which is consistent with the hypothesis that CABYR-A is the only form that binds calcium in sperm. These observations confirmed that, despite the presence of the stop codon in CR-A, splice variants containing CR-B are expressed during spermiogenesis and assemble into the fibrous sheath of the principal piece; however, calcium binding occurs only to those CABYR isoforms containing CABYR-A.