Although sleep is a ubiquitous animal behavior, the molecular mechanism of sleep homeostasis remains unknown. We performed high-throughput screening of ENU-mutagenized mice in order to identify genes regulating sleep/wake behavior. We have so far analyzed EEG/EMG data of more than 6,000 mutagenized male mice. We established several pedigrees showing heritable sleep/wakefulness abnormalities. Among them, the Sleepy mutant pedigree shows 30% reduction in 24-h wake time. To map a chromosomal region responsible for the sleep phenotype of Sleepy mutant mice, we performed a linkage analysis in N2 mice, obtained by backcrossing the mutagenized founder C57BL/6J male to C57BL/6N female mice for two generations. The analysis revealed a single peak with a LOD score of more than 20. Whole exome sequencing of mutants and wild-type littermates from the Sleepy pedigree identified a nucleotide change specific to Sleepy mutant mice within the mapped chromosomal region. The single nucleotide substitution abrogates a splice donor site of the gene that we termed Sleepy. RT-PCR analysis of the brain and liver mRNA found a short variant of Sleepy mRNA specific to Sleepy mutant mice. Functional analyses of the Sleepy gene is now underway.