Cyclin D1 is a cell-cycle regulator essential for G1 phase progression and a candidate proto-oncogene implicated in pathogenesis of several human tumour types, including breast carcinomas. In spite of the accumulating genetic evidence, however, there are no data regarding abundance and properties of the cyclin D1 protein in breast cancer. We now report aberrant nuclear overexpression/accumulation of the cyclin D1 protein in about half of the 170 primary breast carcinoma specimens analyzed by monoclonal antibody immunohistochemistry, indicating that the frequency of cyclin D1 abnormalities may be considerably higher than previously deduced from DNA amplification studies. A comparison of the expression patterns in matched lesions at different stages of tumour progression revealed that the cyclin D1 protein aberration appears to reflect a relatively early event and that, when acquired by a tumour, it is maintained throughout breast cancer progression including metastatic spread. In both tumour tissues and breast cancer cell lines, the abundance of this protein shows characteristic variations consistent with a cell-cycle oscillation and the peak levels expressed in G1. In all 7 cell lines whose retinoblastoma (Rb) protein is mutant or complexed to SV40 T antigen, exceptionally low levels of cyclin D1 protein and mRNA were found. Antibody-mediated and anti-sense oligonucleotide knockout experiments demonstrate the requirement for the cell-cycle regulatory function of cyclin D1 in breast cancer lines with single or multiple copies of the gene and reveal the absence of such a requirement in the cell lines with Rb defects. Our data are consistent with the notion that the emerging "Rb-cyclin D1 pathway" represents a frequent target of oncogenic abnormalities in breast cancer.