Cripto-1 (CR-1), amphiregulin (AR), and heregulin alpha (HRGalpha) are three recently discovered epidermal growth factor (EGF)-related peptides. The expression of these proteins was determined in MCF-7, ZR-75-1, T-47D, SK-BR-3, MDA-MB-231, MDA-MB-468, and Hs-578T human breast cancer cell lines using reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blotting, and immunocytochemistry (ICC). The expression of CR-1 mRNA was detected by RT-PCR in all of the breast cancer cell lines. AR mRNA was detected by Northern blot analysis in MCF-7, ZR-75-1, T-47D, MDA-MB-231, and MDA-MB-468 cells while HRGalpha mRNA was expressed in only MDA-MB-231 and Hs-578T cells. All estrogen receptor-positive cell lines were found to express AR mRNA, and estrogen was able to induce AR mRNA expression in estrogen-depleted MCF-7 cells. CR-1 and AR proteins could be immunocytochemically detected in the breast cancer cell lines that were expressing CR-1 and AR mRNA using monospecific rabbit polyclonal antibodies. The anti-CR-1 antibody was also used to examine 26 human primary breast carcinomas by ICC for CR-1 expression. Seventy-five percent of the carcinomas were found to express the CR-1 protein while the adjacent non-involved breast epithelium was negative. These data demonstrate that CR-1, AR, and HRGalpha are coexpressed in human breast cancer cells and suggest that these three EGF-related peptides might perform a role in the autocrine growth regulation of human breast carcinoma cells.