Mouse sperm possess a phosphotyrosine-containing hexokinase type 1 (HK1) that is associated with the plasma membrane fraction of these cells (Kalab et al., 1994; J. Biol Chem 269:3810-3817). This apparent plasma membrane association appears unique, since somatic HK1 is normally cytoplasmic or bound to the outer mitochondrial membrane via contact sites with a voltage-dependent anion channel (porin) through a porin-binding domain. In male germ cells, three cDNA clones have been described that encode unique HK1 isoforms (HK1-sa, HK1-sb, HK1-sc) that do not contain porin binding domains (Mori et al., 1993: Biol Reprod 49:191-203). This suggests that these proteins might not be localized to the outer mitochondrial membrane and could have alternative functions in germ cells and/or sperm. We demonstrate in the mouse that male germ cells and sperm could potentially express four HK1 isoforms (HK1-sa, HK1-sb, HK1-sc, and the somatic HK1). At the protein level, at least one of the HK1 isoforms becomes phosphorylated on tyrosine residues during spermatogenesis. Treatment of sperm membrane fractions to dissociate the phosphotyrosine-containing HK1 (pY-mHK1) yields results demonstrating that pY-mHK1 has properties of an integral membrane protein. Indirect immunofluorescence using a monoclonal antibody to HK1 demonstrates specific staining both in the head and tail regions of sperm. Surface biotinylation of intact sperm followed by precipitation with either polyclonal HK1 antiserum or with avidin-Sepharose suggests that pY-mHK1 possesses an extracellular domain. These results suggest that mouse sperm contain at least one HK1 isoform that is present on the sperm head, has an extracellular domain, and behaves as an integral membrane protein.