Heregulin-beta1 (HRG) is a regulatory polypeptide having several distinct biological effects in mammary epithelial cells. To address the hypothesis that HRG selectively regulates gene expression, we performed differential display screening using cells grown in the presence or absence of HRG. One cDNA clone upregulated by HRG was identical to human calnexin, a protein with molecular chaperone function. This is the first demonstration of the regulation of calnexin mRNA and protein expression by a physiologically relevant polypeptide factor in human breast cancer cells. HRG stimulation also caused a rapid redistribution of calnexin from vesicle-like structures in the cell cytoplasm to the perinuclear area and to the cell membrane. Furthermore, HRG induced colocalization and physical interaction of calnexin with the HER2 growth factor receptor. Finally, calnexin protein levels were increased in progressive stages of human breast cancer. These findings suggest that stimulation of calnexin expression by HRG may constitute a mechanism of protein redistribution and facilitate downstream signaling events in growth-factor-activated cells.