The purpose of our study was to evaluate the effect of testosterone and its metabolic pathway in MCF-7 cells in culture. Testosterone exhibited a dose-dependent (from 0.1 to 10 nM) and time-dependent (from 3 to 9 days) growth stimulation. The metabolic pathway was investigated following treatment with two testosterone concentrations: one stimulating (10 nM) and one not affecting (0.1 nM) cell growth. Celite column chromatography was used to separate H-3-testosterone metabolites, whose identity was confirmed by gas chromatography-mass spectrometry analysis. The main findings of the metabolic study were: i) recovery of a large amount of untransformed testosterone; ii) a high conversion of testosterone to conjugated, biologically inactive metabolites; iii) the highest level of Sa-diol among the metabolites of testosterone; iv) a conversion (2%) of testosterone into oestradiol, which resulted in a growth stimulatory concentration when testosterone was used at 10 nM. We conclude that in our experimental conditions androgens and oestrogens can concur to stimulate MCF-7 cell growth through androgen receptor-mediated and oestrogen receptor-mediated mechanisms.