The alpha 2u-globulins are the major urinary proteins in adult male rats. They are encoded by a gene family, the expression of which is under multihormonal control in the liver. Glucocorticoids are positive regulatory hormones and we have analyzed the contribution of 5'-upstream sequences to the induction by dexamethasone of two cloned members of the family transfected into mouse L-cells. The results demonstrate that sequences from -762 bp to -226 bp of clone 91 are required for the 24-fold level of induction that was observed. Addition of 5.5 kb of upstream sequence beyond -762 bp did not alter the level of induction significantly, whereas deletion of the DNA between -762 bp and -226 bp reduced inducibility to about 4-fold. Sequencing of this region revealed that an element, 5'-AGAACAggtTTCAAA-3', similar to the 15 bp consensus glucocorticoid response element 5'-AGAACAnnnTGTACC-3', is situated 513 bp upstream of the transcription start site. We infer that this element or its left half site is necessary for the dexamethasone-induced expression of clone 91 from the observation that a second gene, clone 2, that contained a base substitution at position 5 in the left half site was not inducible. It now appears that at least three distinct cis-acting regulatory regions, all of which bind to the glucocorticoid receptor in vitro, may contribute to the full induction of clone 91 by dexamethasone. These are: the distal upstream region identified by this study, a proximal upstream region that binds not only the receptor but also alpha 2uNF1, a constitutively expressed nuclear protein required for induction and a region within the fourth intron that contains five tandem receptor binding sites.