Eye phenotypes were investigated in Le-CreTg/-; Pax6fl/+ mice, which were expected to show tissue-specific reduction of Pax6 in surface ectoderm derivatives. To provide a better comparison with our previous studies of Pax6+/- eye phenotypes, hemizygous Le-CreTg/- and heterozygous Pax6fl/+ mice were crossed onto the CBA/Ca genetic background. After the Le-Cre transgene had been backcrossed to CBA/Ca for seven generations, significant eye abnormalities occurred in some hemizygous Le-CreTg/-; Pax6+/+ controls (without a floxed Pax6fl allele) as well as experimental Le-CreTg/-; Pax6fl/+ mice. However, no abnormalities were seen in Le-Cre-/-; Pax6fl/+ or Le-Cre-/-; Pax6+/+ controls (without the Le-Cre transgene). The severity and frequency of the eye abnormalities in Le-CreTg/-; Pax6+/+ control mice diminished after backcrossing Le-CreTg/- mice to the original FVB/N strain for two generations, showing that the effect was reversible. This genetic background effect suggests that the eye abnormalities are a consequence of an interaction between the Le-Cre transgene and alleles of unknown modifier genes present in certain genetic backgrounds. The abnormalities were also ameliorated by introducing additional Pax6 gene copies on a CBA/Ca background, suggesting involvement of Pax6 depletion in Le-CreTg/-; Pax6+/+ mice rather than direct action of Cre recombinase on cryptic pseudo-loxP sites. One possibility is that expression of Cre recombinase from the Pax6-Le regulatory sequences in the Le-Cre transgene depletes cofactors required for endogenous Pax6 gene expression. Our observation that eye abnormalities can occur in hemizygous Le-CreTg/-; Pax6+/+ mice, in the absence of a floxed allele, demonstrates the importance of including all the relevant genetic controls in Cre-loxP experiments.