In this study we analysed the cytosolic concentrations of the estrogen-regulated protein pS2 in tumors of 462 breast cancer patients, 16 benign breast tumors and 58 metastases. The median pS2 values were highest in breast cancer, followed by benign tumors and metastases (Kruskal-Wallis Test: p < 0.05). Information on other prognostic factors and clinical outcome was available for 354 patients (median follow-up, 35 months). We found a pS2 value of 2 ng/mg protein to be the best cut-off level to discriminate between pS2+ (63%) and pS2- (37%) tumors with respect to relapse-free survival (RFS) and overall survival (OS). The pS2 status was significantly correlated with age, estrogen receptor (ER) and progesterone receptor (PR) status. pS2 was negatively correlated with grading and was more often positive in invasive lobular than in invasive ductal carcinomas. ER, pS2 and grading were highly significantly correlated with each other. In univariate analysis pS2- patients showed a significantly shorter RFS (p = 0.0001) and OS (p = 0.0005). However, multiple regression analysis revealed that in our series of patients the pS2 status provides no independent prognostic information.