This study evaluated Na/K-ATPase alpha 1- and alpha 3-subunit isoform polypeptide expression and localization during bovine preattachment development. Na/K-ATPase cation transport activity from the one-cell to blastocyst stage was also determined by measuring ouabain-sensitive 86Rb+ uptake. Both alpha1- and alpha 3-subunit polypeptides were detected by immunofluorescence to encircle the entire cell margins of each blastomere of inseminated zygotes, cleavage stage embryos, and morulae. Immunofluorescent localization of alpha1-subunit polypeptide in bovine blastocysts revealed an alpha1 immunofluorescence signal confined to the basolateral membrane margins of the trophectoderm and encircling the cell periphery of each inner cell mass (ICM) cell. In contrast, alpha 3-subunit polypeptide immunofluorescence was localized primarily to the apical cell surfaces of the trophectoderm with a reduced signal present in basolateral trophectoderm regions. There was no apparent alpha 3-subunit signal in the ICM. Analysis of 86Rb+ transport in vitro demonstrated ouabain-sensitive activity throughout development from the one-cell to the six- to eight-cell stage of bovine development. 86Rb+ uptake by morulae (day 6 postinsemination) did not vary significantly from uptake detected in cleavage stage embryos; however, a significant increase was measured at the blastocyst stage (P < 0.05). Treatment of embryos with cytochalasin D (5 micrograms/ml) did not influence 86Rb+ uptake in cleavage stage embryos. Cytochalasin D treatment however was associated with a significant rise in ion transport in morulae and blastocysts (13.49 and 61.57 fmol/embryo/min, respectively) compared to untreated controls (2.65 and 22.83 fmol/embryo/min, respectively). Our results, for the first time, demonstrate that multiple Na/K-ATPase alpha-subunit isoforms are distributed throughout the first week of mammalian development and raise the possibility that multiple isozymes of the Na/K-ATPase contribute to blastocyst formation.