Chemical mutagenesis of the mouse is ongoing in several centers around the world, with varying estimates of mutation rate and number of sites mutable to phenotype. To address these questions, we sequenced approximately 9.6 Mb of DNA from G1 progeny of ethylnitrosourea-treated mice in a large, broad-spectrum screen. We identified 10 mutations at eight unique sites, including six nonsynonymous coding substitutions. This calibrates the nucleotide mutation rate for two mutagenesis centers, implies significance criteria for positional cloning efforts, and provides working estimates of effective genetic target sizes for selected phenotypes.