This study demonstrates that acetaminophen (p-acetamidophenol) stimulates proliferation of estrogen-responsive cultured breast cancer cells and assesses if the proliferative activity of p-acetamidophenol is influenced by the -OH moiety position on the benzene ring. The effects of p-, m-, and o-acetamidophenol on cell number and on percentage cells in S phase of the cell cycle were determined for two estrogen receptor positive, human breast cancer cell lines, T47D and MCF7. Therapeutic concentrations of p-acetamidophenol (0.1 mM) significantly increased breast cancer cell proliferation. The relative order of potency of isomers in stimulating proliferation in both cell types was p- > m- > o-acetamidophenol, indicating the -OH position on the benzene ring influences the proliferation output in cultured breast cancer cells.