We have investigated the contribution of the tight junction (TJ) transmembrane protein junction-adhesion-molecule 1 (JAM-1) to trophectoderm epithelial differentiation in the mouse embryo. JAM-1-encoding mRNA is expressed early from the embryonic genome and is detectable as protein from the eight-cell stage. Immunofluorescence confocal analysis of staged embryos and synchronized cell clusters revealed JAM-1 recruitment to cell contact sites occurred predominantly during the first hour after division to the eight-cell stage, earlier than any other TJ protein analysed to date in this model and before E-cadherin adhesion and cell polarization. During embryo compaction later in the fourth cell cycle, JAM-1 localized transiently yet precisely to the apical microvillous pole, where protein kinase Czeta (PKCzeta) and PKCdelta are also found, indicating a role in cell surface reorganization and polarization. Subsequently, in morulae and blastocysts, JAM-1 is distributed ubiquitously at cell contact sites within the embryo but is concentrated within the trophectoderm apicolateral junctional complex, a pattern resembling that of E-cadherin and nectin-2. However, treatment of embryos with anti-JAM-1-neutralizing antibodies indicated that JAM-1 did not contribute to global embryo compaction and adhesion but rather regulated the timing of blastocoel cavity formation dependent upon establishment of the trophectoderm TJ paracellular seal.