The role of the adhesion molecule CD44 in the natural history of breast cancer is controversial. We investigated the CD44 isoform CD44v5 and CD44v6 concentrations in the cytosol of 90 breast cancer specimens, 9 fibroadenomas and 22 normal breast tissue specimens by means of ELISA. CD44v5 and CD44v6 cytosol concentrations were statistically significantly higher in breast cancer compared with fibroadenoma and normal breast tissue (Mann-Whitney U-test, p = 0.009 and p < 0.001, respectively). When CD44 isoforms were correlated with lymph node involvement, histological grading, histological type, tumor stage and age at diagnosis, we found no statistically significant correlation with any of the investigated clinico-pathological parameters. In univariate and multivariate analyses, CD44v5 and CD44v6 were of no prognostic relevance regarding disease-free survival in breast cancer patients (log-rank test, p = 0.16 and p = 0.08, respectively). Our results indicate that CD44 isoforms are increased in samples from tumors relative to normal tissue. Our data show that CD44v5 and CD44v6 isoform expression, although up-regulated by malignant transformation, is not required to acquire a metastatic phenotype in breast cancer. Furthermore, our data support the assumption that cytosolic CD44 isoforms are of no prognostic relevance for disease-free survival of breast cancer patients.