The objective of this study was to establish a method by which trophectodermal cells originating from individual preimplantation bovine embryos could be perpetuated in monolayer culture. A single, Day-11 bovine embryo collected nonsurgically from a mixed-breed beef cow was cultured in Ham's F10 medium supplemented with fetal bovine serum, sodium pyruvate, insulin, and epidermal growth factor. After 13 d in culture the embryo had adhered to the surface of the plastic culture vessel and a monolayer covering 0.3 cm2 had developed in the manner of a tissue explant. The monolayer was successfully dispersed using trypsin-EDTA and the cells were passaged. Expansion to a 25-cm2 flask was achieved by the 4th passage. By passaging cultures at a dilution ratio of 1:2, cells were maintained for 38 passages before growth slowed. Transfers beyond the 44th passage were unsuccessful. The cell line, designated BE-13, was successfully frozen and thawed at the 9th, 12th, 15th, and 20th passages. The cell line contains both mono- and binucleate cells with a prominent rough endoplasmic reticulum characteristic of ruminant trophoblast cells. Susceptibility to eight bovine viruses was demonstrated. Such cell lines may provide inexpensive systems for the study of trophoblast metabolism and for investigation of the role of the trophoblast in the pathogenesis of selected bovine abortifacient diseases. Because of their range of viral susceptibility, these cells might also be useful for diagnostic purposes.