In the field of rat genomics and transgenics, efforts to produce germline rat ES cells have been made for over nineteen years. Authentic rat ES cells were described for the first time in December 2008. Proven germline rat ES cells are needed if this breakthrough is to impact the field. We derived ES cells 2i medium from 1) outbred Long-Evans (Crl:LE), 2) inbred hybrid (DA/OlaHsd X BN/NHsdMcwi)F1 and 3) hybrid (F344/Hsd X BN/NHsdMcwi)F1 rat blastocysts. Outbred Crl:LE hooded rats were selected to take advantage of coat color differences when ES cells are combined with albino F344/NHsd blastocysts to produce ES cell-rat chimeras. Hybrid rat ES cell lines were developed with BN/NHsdMcwi so as to take advantage of the public rat sequence and genomic library resources for the construction of isogenic gene targeting vectors. Hybrid F1 rat ES cell lines are also expected to show robust contribution to ES cell-rat chimeras. Gene targeting vectors designed to produce null alleles were constructed by recombineering technology in bacterial artificial chromosomes (BACs) from the CHORI-230 BAC library. BN/NHsdMcwi genomic DNA was used to construct the library that is the source of the public rat genome sequence. It is well known that genetic identity between cloned gene targeting vectors and chromosomal sequences enhance the efficiency of homologous recombination mediated changes. The exact sequence identity between targeting vectors and the genome of the hybrid F1 rat ES cells is expected to maximize the frequency of homologous recombination compared to Crl:LE ES cells. Rat ES cell line derivation exceeds typical mouse ES cell derivation efficiency. We derived 32 rat ES cell lines from 69 donor blastocysts (46% yield) with twice the efficiency of deriving 93 mouse ES lines from 363 blastocysts (26% yield). Results of ES cell-rat chimera production will be reported. The advent of germline rat embryonic stem (ES) cell lines ushers in a new era in rat model development. Successful proof-of-principle projects will bolster the value of rat models to address biomedical research questions with rat models.