Accumulation of hyaluronan (HA) in pericellular stroma and carcinoma cells is predictive of unfavorable patient prognosis in many epithelial cancers. However, it is not known whether the HA originates from carcinoma or stromal cells, or whether increased expression of hyaluronan synthase proteins (HAS1-3) contributes to HA accumulation. In this study, localization and expression of HAS1-3 were evaluated immunohistochemically in 278 cases of human breast cancer, and correlated with prognostic factors and patient outcome. Both carcinoma cells and stromal cells were HAS-positive. In carcinoma cells, HAS1 and HA stainings correlated with each other, and HAS1 associated with estrogen receptor negativity, HER2 positivity, high relapse rate, and short overall survival. In stromal cells, the staining levels of all HAS isoforms correlated with the stromal HA staining, stromal cell CD44, high relapse rate, and short overall survival of the patients. In addition, expression levels of stromal HAS1 and HAS2 were related to obesity, large tumor size, lymph node positivity, and estrogen receptor negativity. Thus, stromal HAS1 and HAS3 were independent prognostic factors in the multivariate analysis. The data suggest that increased levels of HAS enzymes contribute to the accumulation of HA in breast cancer, and that HA is synthesized in carcinoma cells and stromal cells. The study also indicates that HAS enzyme levels are related to tumor aggressiveness and poor patient outcome representing potential targets for therapy.