A total of 14 microsurgically produced zona pellucida-free bovine demi-blastocysts were cultured for 3 days in tissue culture medium (TCM) 199 supplemented with 10% heat-inactivated newborn calf serum (NBCS). Developing embryos were continuously cultured in TCM 199 plus 10% NBCS on a feeder-layer of murine embryonic fibroblasts, that had been incubated with mitomycin C (10 ~tg/ml) for 3 h prior to the onset of embryo cultivation to block mitotic activity of the fibroblasts. After 2 days, 3 expanded blastocysts were attached to the feeder-layer and both trophoblastic cells and inner cell mass (ICM) cells became apparent on the 9th day of culture in 2 out of the 3 expanded blastocysts. Five days later, the ICM cells were disaggregated by a short-term trypsin treatment. The resulting dissociated clumps were seeded on a new murine embryonic fibroblast feeder-layer and covered with modified minimum essential medium (MEM)-Alpha with 10% fetal calf serum (FCS), 0.1 mM mercaptoethanol, 4.5 g/1 glucose and 20 mM HEPES-buffer (= passage 0). To prevent differentiation of the cells, approximately 1/3 of the MEM-Alpha was replaced by MEM previously incubated on cell line 5637 containing leucaemia inhibitory factor (LIF) for 3 days. Colonies of embryonic stem cell (ES)-like cells were observed 5 days after the 1st passage. These colonies were repeatedly passaged at approximately 2-week intervals. Two bovine ES-like cell lines were established, which grew considerably slower than murine ES cells, but were lost after the 4th passage, possibly because of toxic effects of a new FCS batch. After cytogenetic analysis, 16 out of 18 metaphase plates contained an euploid number of chromosomes with 2 X-chromosomes and 58 autosomes. Distribution of G-