The aim of this study was to examine the cellular distribution of osteopontin (OPN) protein [by immunohistochemical (IHC) analysis] and mRNA [by in situ hybridization (ISH)] in the primary tumors of lymph node negative (LNN) breast cancer patients and to determine whether the level of immunodetectable OPN may be associated with tumor aggressiveness. We examined OPN levels in tumors from 154 patients with LNN breast cancer who were followed for a median of 7 years (range 1.7-16.3 years). IHC staining for OPN was seen in tumor infiltrating macrophages and lymphocytes in 70% of these tumors, and in the carcinoma cells themselves in 26%. ISH was performed to determine cellular distribution of OPN mRNA expression in sections from selected tumors. OPN mRNA was detected in groups of tumor cells, individual tumor cells and tumor infiltrating macrophages and lymphocytes. Matched sections showed that some tumor cells with IHC staining for OPN protein were also positive for OPN mRNA by ISH, in contrast with previous studies which have shown OPN mRNA expression only in tumor infiltrating inflammatory cells. Our results thus indicate that OPN protein can be produced by breast cancer cells in vivo and suggest that it may also be taken up from the environment (i.e., secreted by inflammatory cells or other tumor cells). Tumor cell IHC staining intensity was then assessed using a semiquantitative scoring system. Univariate analysis showed tumor cell OPN positivity above an optimized cutpoint to be significantly associated with decreased disease-free survival (DFS) and overall survival (OS). The results of this pilot study thus suggest that the ability of breast cancer cells to either synthesize OPN or to bind and sequester OPN from the microenvironment may be associated with tumor aggressiveness and poor prognosis.