Treating mice with ethylnitrosourea (ENU) is an efficient means for mutagenizing spermatogonial cells, and this treatment has been proven effective in a variety of screens for both dominant and recessive mutations. However, a significant problem for this technology is that the efficiency of mutagenesis is assessed most often by the empiric determination of a per-locus mutation frequency by using the specific locus test, which is expensive, time-consuming, and logistically difficult. To approach this question more directly and more efficiently, one can utilize methods of PCR-based mutation detection for the characterization of progeny of mutagenized mice. Since this analysis can be done after a single generation of breeding, it is useful as a rapid means for the assessment of the efficiency of mutagen treatment. Furthermore, it is readily imaginable that this strategy can be applied for the general determination of gene function in a systematic manner. Theoretical considerations and empirical analysis suggest that the per-base mutation frequency for a fractionated-dose treatment protocol is on the order of 1 sequence change per 10(5) bp.