Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tailand tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.