DNA cloned in yeast artificial chromosomes (YACs) is a valuable resource for functional experiments in cell culture as well as whole animal systems. Where the size or chimerism of a YAC clone are limiting factors it may be desirable to generate recombinant YAC clones. One such approach is based on mitotic recombination, and we describe the development of a methodology that allows multiple recombination cycles for serial reconstruction of overlapping YACs. This approach employs retrofitting with standard plasmid vectors, transfer of YACs to a common haploid host by Kar1 mating, and selection for recombination with 5-fluoro-orotic acid.