Here, we demonstrate long-term cultivation of alkaline phosphatase- positive rat embryonic stem-like (RES) cell lines. RES cells were characterized by their typical growth in highly compacted cell clusters, which were found to be sensitive against enzymatic dissociation. RES cells expressed stage-specific embryonic antigen-1 (SSEA-1) and transcription factor Oct-4, but Oct-4 mRNA was detected at lower levels compared to mouse ES cells. Once established to tissue culture, RES cells were able to grow in the absence of feeder cells under clonal conditions. Cytokines of the interleukin-6 family known to maintain the undifferentiated state of mouse ES cells were comparatively analyzed for their capacity to maintain the undifferentiated growth of two cell lines, RES-1 and RES-15, in a clonal assay. Rat ciliary neurotrophic factor (rCNTF), human oncostatin M (hOSM), and interleukin-6 and soluble interleukin-6 receptor (IL-6/sIL-6R) were found to support clonal growth of RES cells, but the cytokines did not reach the efficiency of the colony forming ability of leukemia inhibitory factor (LIF). When RES-1 and RES-15 cells were cultivated without feeder cells, SSEA-1 expression was maintained after clonal growth in the presence of LIF and LIF + rCNTF, respectively. Oct-4 mRNA was significantly detected in RES-15 cells when cultivated in the absence of feeder cells in media substituted by LIF and/or IL-6/sIL-6R, as well as without cytokines. In summary, rat embryonic stem-like cell lines could be established from rat blastocysts and were able to proliferate as undifferentiated alkaline phosphatase-positive cells. Embryonal stem cell properties, such as SSEA-1 and Oct-4 expression, were maintained by members of the IL-6 family of cytokines, but most significantly by LIF.