Heregulin (HRG) and 17beta-estradiol (E2) interactions that modulate growth of breast cancer cell lines have recently been demonstrated. We examined the ability of heregulin beta1 (HRGbeta1) and 17beta-estradiol to modulate the biological behavior of estrogen receptor (ER) negative human breast cancer cell lines (AU-565). The proliferation of AU-565, MBA-MB231, and SKBR3 cells was additively inhibited by treatment with 17beta-estradiol (10(-6) M) and HRGbeta1 (10 ng/ml). 17-beta estradiol did not support the transcriptional activation of a reporter gene construct containing an estrogen response element transfected into AU-565 cells. This finding suggested functional endogenous ER was not present in AU-565 cells. However, the cells contained a high number of low affinity estrogen binding sites. 17beta-estradiol only slightly decreased basal tyrosine phosphorylation of ErbB-2 and ErbB-3. Estrogen and HRGbeta1 treatment resulted in an increase of c-myc mRNA. We conclude that 17beta-estradiol and HRGbeta1, in combination, potently inhibit cell proliferation of three ER negative breast carcinoma cell lines.