Identification of the genes involved in the development of the mouse inner ear and developmental studies of mice that bear mutations in these genes is an important approach to understanding genetically determined human auditory dysfunction. Towards this end, we initiated a gene trap screen designed to simultaneously mark and mutate genes in mouse embryonic stem cells by the insertion of a lacZ reporter gene. Expression of beta-galactosidase in gene trap cell lines was monitored both before and after the addition of factors that are known to affect inner ear development. Gene trap cell lines that expressed beta-galactosidase under one or more culture conditions were used to create chimeric mouse embryos for studies of reporter gene expression in vivo. A high proportion of these gene trap insertions were expressed in the developing inner ear, suggesting that this strategy provides an effective means of identifying genes that may be involved in inner ear development or function.