A simple and accurate analysis system for the estimation of recombination efficiency in vivo using fluorescence-activated cell sorting (FACS) was designed and was subsequently used to compare the efficiency of recombination related to different spacer mutants. F-3 and F-5 mutant sequences were used for Flpe-mediated cassette exchange, and m2 and lox2272 mutant sequences were used for Cre-mediated cassette exchange due to their high incompatibility with wild-type sequences. The incompatibilities with wild-type were almost the same between mutant sequences. However, the recombination efficiencies were different. F-3 and m2 could mediate more efficient recombination than F-5 and lox2272, respectively. These results are consistent with the fact that the sequence of spacer region affects not only the reactivity upon wild-type sequence but also the recombination efficiency. It was also confirmed that the recombination process was mediated in a site-specific manner through PCR analysis using different sizes of exchange cassettes. In this experiment, the feasibility of the FACS analysis system for the estimation of recombination efficiency was verified. This system should be readily applicable for estimating recombination efficiency of other various mutant candidates, which will contribute to more precise and efficient site-specific recombination. (c) 2006 Elsevier B.V. All rights reserved.