To develop an systematic in vitro approach for the study of genomic imprinting, we generated a new library of human/mouse A9 monochromosomal hybrids. We used whole cell fusion and microcell-mediated chromosome transfer to generate A9 hybrids containing a single, intact, bsr-tagged human chromosome derived from primary fibroblasts. A9 hybrids were identified that contained either human chromosome 1, 2, 4, 5, 7, 8, 10, 11, 15, 18, 20, or X. The parental origin of these chromosomes was determined by polymorphic analysis using microsatellite markers, and matched hybrids containing maternal and paternal chromosomes were identified for chromosomes 5, 10, 11 and 15. The imprinted gene KVLQT1 on human chromosome 11p15.5 was expressed exclusively from the maternal chromosome in A9 hybrids, and the parental-origin-specific expression patterns of several other imprinted genes were also maintained. This library of human monochromosomal hybrids is a valuable resource for the mapping and cloning of human genes and is a novel in vitro system for the screening of imprinted genes and for their functional analysis.