Chicken cell lines derived from avian leukosis virus-induced B-cell tumors are highly recombination-proficient. In these cells, homologous recombination between transfected DNA sequences and the corresponding chromosomal alleles occurs at high frequency, and targeting efficiencies of 10-90% can be obtained. We previously described a chromosome shuttle approach in which these cells can be used to modify human chromosomal alleles at high efficiency. In this method, marked human chromosomes are transferred into the chicken B-cell line, DT40, by microcell fusion. Human loci on the introduced chromosome can be modified efficiently by homologous recombination in the DT40/human microcell hybrids. The modified human chromosomes can then be transferred back to mammalian recipient cells for functional tests. In this report, we describe procedures for performing interclass microcell transfers between mammalian donor cells and DT40 recipients. We also describe transfection procedures for DT40/human microcell hybrids, which allow the recovery of transfectant clones containing specific homologous modifications. Finally, methods for transferring the modified human chromosomes from DT40/human microcell hybrids back to mammalian recipients are discussed.