Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 tm1Ued ( Pax6 fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 fl/fl and heterozygous Pax6 fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 Sey- Neu ( Pax6 −) null allele. Pax6 fl/− compound heterozygotes had more severe eye abnormalities than Pax6 + /− heterozygotes, implying that Pax6 fl differs from the wild-type Pax6 + allele. Immunohistochemistry showed that the Pax6 fl/− corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.