The Mr 24,000 and Mr 52,000 estrogen-regulated cytosol proteins, and the breast cancer-associated antigen DF3 have been studied in an immunocytochemical assay. Primary tumor specimens from 119 patients with advanced breast cancer who received endocrine therapy have been studied. Monoclonal antibodies were used for the detection of the proteins in formalin-fixed paraffin-embedded blocks. No correlation between Mr 52,000-positive specimens and the presence of estrogen receptor (ER) could be established (p = 0.87, chi-square test) whereas a statistically significant association between Mr 24,000 (p = 0.0002), DF3 antigen (p = 0.044) and ER was demonstrated. No intercorrelation was found between Mr 24,000 and Mr 52,000 or DF3 (p = 0.63, 0.98 and 0.12 respectively). Clinical response was evaluated for immunocytochemical findings, Mr 24,000 (p = 0.37), Mr 52,000 (p = 0.61) and DF3 (p = 0.68) showed no association whereas ER was statistically correlated (p = 0.00005). Neither overall survival nor disease-free survival correlated to Mr 24,000 (p = 0.18 and 0.75 respectively, logrank test), Mr 52,000 (p = 0.095 and 0.38), or DF3 (p = 0.22 and 0.13) staining, whereas ER-positive tumors did (p = 0.00005). Discrimination between ER-positive responders and ER-positive non-responders was not possible using either Mr 52,000, Mr 24,000 or DF3 staining. Based on our findings we conclude that immunocytochemical staining for Mr 52,000, Mr 24,000 or DF3 cannot be used as a marker to predict response to endocrine therapy in patients with advanced or recurrent breast cancer.