Calreticulin is a multifunctional protein that acts as a major Ca(2+)-binding (storage) protein in the lumen of the endoplasmic reticulum. It is also found in the nucleus, suggesting that it may have a role in transcription regulation. Calreticulin has been reported to bind to the synthetic peptide KLGFFKR, which is almost identical to an amino-acid sequence in the DNA-binding domain of the superfamily of nuclear receptors. Could calreticulin interact with the DNA-binding domain of these receptors and affect their function? Here we report that the amino terminus of calreticulin interacts with the DNA-binding domain of the glucocorticoid receptor and prevents the receptor from binding to its specific glucocorticoid response element. Overexpression of calreticulin in mouse L fibroblasts inhibits glucocorticoid-response-mediated transcriptional activation of a glucocorticoid-sensitive reporter gene and of the endogenous, glucocorticoid-sensitive gene encoding cytochrome P450. Together these results indicate that calreticulin may be important in gene transcription, regulating the glucocorticoid receptor and perhaps other members of the super-family of nuclear receptors.