Embryonic stem (ES) cell lines have been derived from the inner cell mass of day 3.5 blastocysts of the inbred mouse strain BALB/cJ. Twenty-three lines were karyotyped and three were selected for injection into C57BL/6J host blastocysts. Two of the three lines, BALB/c-I and BALB/c-IV, produced germ-line chimaeras. The suitability of the BALB/c-I line for gene targeting experiments was tested by transfecting a targeting construct for the interleukin-4 (IL-4) gene. Transfected BALB/c-I cells exhibited efficient homologous recombination of the targeting vector and transmitted the induced mutation through the germline. This newly-characterized BALB/c-ES cell line thus provides an alternative to the traditional 129-derived and the recently described C57BL/6 embryonic stem cell lines, and will be useful in disrupting genes involved in the immune system. Furthermore, the genetically pure BALB/c IL-4 deficient mice will aid in studying the role of IL-4 in several infectious disease models in which the BALB/c mouse is a susceptible strain.