Expression and activity of the Na-K-ATPase within the basolateral membrane domains of the trophectoderm epithelium provide the driving force for accumulation of Na(+) and Cl(-) across the nascent epithelium, mediating fluid movement into the forming blastocoel. Within the trophectoderm of the bovine blastocyst, multiple isozymes of the Na-K-ATPase are expressed. Immunolocalization has demonstrated that the alpha1-isozyme localizes within the basolateral membrane, whereas the alpha 3-isozyme localizes to the apical cell margins. Gene-specific RT-PCR and wholemount indirect immunofluorescence confocal laser scanning microscopy were used to examine expression of the Na-K-ATPase gamma-subunit (a regulatory subunit of the Na-K-ATPase) throughout development of bovine preattachment embryos in vitro. Expression of mRNA transcripts for the gamma-subunit was detected throughout bovine pre-attachment development from the fertilized one-cell embryo to the blastocyst stage. A similar pattern of expression was also observed for gamma-subunit protein, and immunofluorescence was detected within the membranes of embryonic blastomeres at all stages of development. In contrast to the expression patterns observed for the alpha-subunits, gamma-subunit proteins were detected in both the basolateral and apical cell margins of the trophectoderm, and surrounding all cells of the inner cell mass. Co-localization studies demonstrated that gamma-subunit peptides are co-expressed with the alpha1-subunit in the basolateral domains of the trophectoderm. These results indicate a role for the gamma-subunit of the Na-K-ATPase in modulating Na(+)-pump activity in both apical and basolateral margins of the trophectoderm during formation and expansion of the bovine blastocyst, and adds a further level of complexity to Na(+)-pump regulation of cavitation.