Caveolin-1 belongs to a family of scaffolding proteins, and the P132L point mutation of this gene has been found in up to 16% of all examined breast cancers. Subsequent studies have revealed that the P132L mutation exerts a dominant negative effect through misfolding during caveolin-1 oligomerization. However, this mutation has not been validated in other series of breast cancer samples. Contradictory to the suggested tumor suppressor function, overexpression of caveolin-1 is common in various cancer types. To clarify these inconsistent results, we examined the caveolin-1 mutation in a large series of breast cancer specimens. We first used a standard direct sequencing method and found that none of the 99 breast cancers tested had this mutation. Then we developed a sensitive method for a paraffin section that could detect the mutant allele at a rate of as little as 0.1% among wild-type allele copies. Even when using this sensitive method, none of the 80 estrogen receptor-positive breast tumors had the P132L mutation. Furthermore, 270 cancers in various organs were examined, and no caveolin-1 mutations were detected. These results raise doubt about the presence of the caveolin-1 P132L mutation in breast cancer and other cancer types, and thus further studies are warranted.