Canine embryonic stem (cES) cell lines were generated to establish a large-animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell-derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12-16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13-14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)-7, has been maintained through 34 passages and is presented here. FHDO-7 cells are alkaline phosphatase-positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miRs) (miR-302b, miR-302c, and miR-367) characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm-derived neuronal cells expressing gamma-enolase and beta 3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing alpha-fetoprotein or Clara cell-specific protein.