Gli2 and Gli3 respond to the Hedgehog (Hh) signal in mammals by undergoing posttranslational modifications and moving to the nucleus. The study of Gli proteins has been hampered by the fact that their overexpression in cells prevents their proper regulation. To address this issue, we have developed a method of rapid generation of stable cell lines expressing near-endogenous and approximately equal levels of wild-type and mutant Gli proteins. This method is applicable to the study of effects of various mutations on Gli protein modifications and activity.