The cytosol values of estrogen-regulated proteins cathepsin D and pS2 were evaluated in 166 patients with breast diseases by a receiver-operating characteristic (ROC) curve analysis. Our aim was to compare their diagnostic accuracy and to define optimal cut-off levels for the cytosol values of these proteins in breast cancer (n = 133). The preoperative mean cytosol cathepsin D values in the breast cancer group were higher than in patients with benign breast disease (p < 0.001). The correlation coefficient (Pearson's r) between Cathepsin D and pS2 measurements was 0.286 (p < 0.001) in breast cancer patients. The ROC analysis indicated that the cathepsin D test was more sensitive than the pS2 test at all specificity levels, and the cathepsin D test reached considerably high sensitivity (86%) at the specificity level of 80%. According to present study, it would seem optimal to use the cut-off level of 33.0 pmol/mg for cathepsin D analysis and the level of 31.0 ng/mg for pS2 in breast cancer. In conclusion, the results of ROC curve analysis suggest that the cathepsin D test has advantage over pS2 because of its higher sensitivity in breast cancer.