Fundamental to placental morphogenesis is union between the allantois and the chorion, two tissues initially separated in the conceptus. Results of previous studies in the mouse have suggested that chorio-allantoic union is driven by the developmental maturity of the allantois and involves molecular interactions between Vascular Cell Adhesion Molecule (VCAM-1) in the allantois and alpha4-integrin in the chorion. Little more is known about the cellular and/or molecular control of this important morphogenetic event in any species.Gross, histological, microsurgical and immunohistochemical analyses in the mouse conceptus revealed that placental ontogeny took place in three major steps. The first, chorio-allantoic contact, was not enduring and was mediated by the allantois' mesothelial surface and the mesodermal component of the chorion. Modest amounts of VCAM-1 were found in distal allantoic mesothelium, whilst levels of alpha4-integrin were high throughout chorionic mesoderm. The second step, chorio-allantoic fusion, was more enduring. During this time, the distal allantoic region contained maximal levels of VCAM-1, and all allantoises had expanded far enough to reach the posterior chorion from where they spread toward a central chorionic depression. The last step, breakdown of chorio-allantoic fusing surfaces, was dependent upon chorio-allantoic fusion and resulted in the intimate juxtaposition of allantoic endothelium and chorionic ectoderm, possibly as a result of VCAM-1-mediated interactions. The umbilical connection was thereafter fixed at its perimeter to the chorionic surface by large amounts of VCAM-1 in disto-lateral allantoic mesothelium and alpha4-integrin in the remaining peripheral mesodermal component of the chorion.Thus, chorio-allantoic union is highly regulated, taking place in multiple steps. It is dependent upon the developmental maturity of distal allantoic mesothelium and involves the mesodermal component of the chorion. Breakdown of fusing surfaces enables penetration of the allantoic vasculature into the chorion. These findings provide a secure developmental foundation in which to elucidate the genetic control of early placentation.