The Cre/ loxP recombination system is a commonly used tool to alter the mouse genome in a conditional manner by deletion or inversion of loxP ‐flanked DNA segments. While Cre‐mediated deletion is essentially unidirectional, inversion is reversible and therefore does not allow the stable alteration of gene function in cells that constitutively express Cre. Site‐directed mutagenesis yielded a pair of asymmetric loxP sites ( lox66 and lox71 ) that display a favorable forward reaction equilibrium. Here, we demonstrate that lox66 / lox71 mediates efficient and predominantly unidirectional inversion of a switch substrate targeted to the mouse genome in combination with either inducible or cell type‐specific cre ‐transgenes in vivo .