Given that thousands of genes exist in the mammalian genome, criteria are needed to prioritize their functional analysis and to decrease the likelihood of producing gene‐targeted mice that lack overt phenotypes. Initial analysis efforts are likely to be fruitful if focused on genes encoding large proteins, since at least some large proteins serve as frameworks for intricate assembly of protein complexes, and their inactivation would render definitive, observable phenotypes. Here, we describe the functional characterization of the murine homologues of five human KIAA genes (KIAA1409, KIAA1440, KIAA1447, KIAA1768, and KIAA1276) that encode large proteins. Gene‐targeted mice had phenotypic and developmental defects resulting from the functional deletion of three of these five genes. Mice with targeted disruption of KIAA1409 lacked the ability to drink, and those with targeted disruption of KIAA1447 displayed hind leg motor dysfunction. Disruption of KIAA1440 led to embryonic lethality at the blastocyst stage. The high success rate of our approach demonstrates the rationale for the genome‐wide functional examination of large proteins in mice using reverse genetics.—Nakayama, M., Iida, M., Koseki, H., Ohara, O. A gene‐targeting approach for functional characterization of KIAA genes encoding extremely large proteins. FASEB J. 20, E1061–E1070 (2006)