Amplification and/or increased expression of cyclin D1 occurs in an appreciable fraction of primary human breast carcinomas and several other types of human cancer. In addition, overexpression of cyclin D1 in rodent fibroblasts enhances growth and malignant transformation. The present study demonstrates that the extent of amplification and expression of cyclin D1 varies widely amongst a series of cell lines established from normal human mammary epithelium or human breast carcinomas. The HBL-100 mammary epithelial cell line did not display amplification or increased expression of cyclin D1. We used retrovirus-mediated transduction to obtain derivatives of this cell line that stably expressed relatively high levels of an exogenous cyclin D1 cDNA. These derivatives displayed an increased doubling time, decreased saturation density, decreased cloning efficiency, decreased anchorage-independent growth, an increased fraction of cells in the S-phase, and decreased tumorigenicity. Thus, increased expression of cyclin D1 in this cell line markedly inhibits rather than enhances growth, which may be due to the prolongation of S-phase.