To compare the effects of the food toxin 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and estradiol in hormone-responsive MCF-7 cells, the cells were exposed to different concentrations of either PhIP or estradiol. The effect of various culture conditions (e.g. phenol red, FBS, vehicle (DMSO/EtOH) and seeding density) on responses was studied. Cells were continuously grown with steroid-containing or -deprived medium, or switched from steroid-containing to -deprived medium for the experiments to minimize the effect of background estrogenicity. Effects of PhIP and estradiol on cell viability and proliferation were determined by ATP analysis and Ki-67 immunocytochemistry. Expression of estrogen receptor alpha, cell stress markers (p53 and ERK) and estrogen responsive proteins (c-myc and ERK) were immunoblotted. All concentrations of estradiol induced cell proliferation, viability and changes in protein expression, typical for estrogenic responses. PhIP, however, increased viability only at low concentrations and depending on culture conditions. No changes in protein expressions by PhIP were noted, not even when switching cells from steroid-containing to -deprived medium which down-regulated the expression of proteins at basal level. Vehicle affected significantly viability, especially after exposure to PhIP, but not protein expression while medium changes affected both. In conclusion, the effects of PhIP and estradiol in MCF-7 cells are dependent on culture conditions. The detected PhIP-induced changes are weaker compared to those induced by estradiol.