Mammalian species are well known to differ in their sensitivity to glucocorticoids, but the molecular basis for this difference remains largely uncharacterized. To address this issue, the transcriptional activity of the mouse and human glucocorticoid receptor (GR) was analyzed on two model glucocorticoid-responsive promoters. Mouse GR (mGR) displayed unique promoter discrimination in response to a range of glucocorticoids, with enhanced activity on a simple glucocorticoid response element (GRE)-based promoter and diminished activity on the complex mouse mammary tumor virus promoter compared with human GR (hGR). Promoter discrimination between mGR and hGR was mapped to a single amino acid change at residue 437 (glycine to valine) of mGR and to sequence differences within individual GREs of the different promoters. Mouse GR displayed higher activation on GREs with a guanine rather than a thymine at the -6 position. Binding studies indicated mGR (mGR437V) displayed a weaker affinity for GREs containing a thymine at the -6 position than a mGR mutant containing a glycine at residue 437 (mGR437G). Despite distinct transcriptional activities, both receptors had similar affinities for response elements that contain a guanine at the -6 position. Our findings support a model by which the presence of a valine residue at position 437 of mGR induces a conformational change that leads to alterations in affinity and/or transcriptional activation in a promoter-dependent context.