Caveolin-1 (Cav-1) mutations, such as P132L, are associated with ER-positive human breast cancers. However, no immunohistochemical methods have yet been described to predict the presence of Cav-1 mutations in human breast cancer. Since the P132L mutation acts in a dominant-negative fashion and causes the mis-localization of Cav-1 in cultured cells in vitro, we hypothesized that of patients carrying this mutation would show a similar Cav-1 staining pattern in vivo. Indeed, while performing histological analysis of Cav-1 immunostaining on human breast cancer samples, we noted the emergence of two distinct epithelial staining patterns: (1) punctate peri-nuclear "Golgi-like" localization; or (2) diffuse cytoplasmic staining. The punctate peri-nuclear staining pattern was associated with ER-alpha positivity and was present mainly in well-differentiated samples. In striking contrast, the diffuse staining pattern was present in poorly differentiated samples, and was not associated with ER-status. DNA sequence analysis revealed that only well-differentiated samples with a punctate staining pattern harbored the Cav-1 P132L mutation. Thus, immunostaining of Cav-1 can be used as a first step to stratify human breast cancer patients and to predict the presence of Cav-1 mutations. As such, the punctate Cav-1 immunostaining pattern can now be used as a screening tool to select patients for Cav-1 mutational analysis.