We examined, by immunohistochemical analysis, the expression of aromatase and estrone sulfatase (E1-STS) which are the two major enzymes involved in in situ estrogen synthesis in breast cancer tissue. In the 83 cases examined, E1-STS, which hydrolyses estrone sulfate (EIS) to estrone (E1), was dominantly detected in tumor cells in 43 (59.0%) cases. Aromatase, which converts androgens to estrogens, was dominantly detected in stromal cells of the tumor. It was detected in 39 (47.0%) of the 83 cases. There was no significant correlation between the expression of these two enzymes and clinicopathological factors. We found a tendency for a correlation between aromatase expression and E1-STS expression in breast tumor (p = 0.075). In terms of the possible use of these enzymes as prognostic indicators, patients who had aromatase in their tumor showed longer relapse-free survival than those lacking aromatase (p = 0.045). Significant correlations between the expression of aromatase and the angiogenesis regulators, vascular endothelial growth factor and thymidine phosphorylase, were found (p = 0.047 and p = 0.046, respectively), though the presence of aromatase did not correlate with intratumoral microvessel density. This may indicate that aromatase is involved in vascular permeability and recruitment of endothelial cells rather than neovascularization. It may be useful to study the expression of aromatase and E1-STS using immunocytochemical analysis for understanding the tumor characteristics in breast cancer.