Protein ubiquitination has been implicated in ATP-dependent protein turnover and normal cell proliferation. To investigate whether the ubiquitin-mediated system is functionally involved in the cancerous state, we examined changes in protein ubiquitination in 52 surgically resected primary breast tumors. Immunohistochemically, ubiquitin (Ub) was identified in the cytoplasm of cancer cells, which were stained more strongly than adjacent normal ductal epithelium. Corresponding immunoblot analysis of normal and neoplastic regions of human breast showed that the immunoreaction for Ub at about 43 kDa was increased in all of the tumors (100%), regardless of the clinical stage or histologic grade. This protein, which gave a single spot on two-dimensional gel electrophoresis, had partial amino acid sequences which were identical to those of actin family members. Our results suggest that ubiquitination of this 43-kDa protein may be involved in the carcinogenesis or biological characteristics of human breast neoplasms.