Invasive lobular carcinoma comprises approximately 10% of human mammary cancers, yet little is known about the molecular basis of this carcinoma. Because cyclin D1 plays a role in the pathogenesis of breast carcinomas of the ductal type, we hypothesized that this confirmed oncogene might also participate in the development of lobular carcinomas. We sought to determine the frequency of cyclin D1 protein overexpression in invasive lobular carcinoma, to investigate the cause of the protein accumulation, and to identify the effects of high levels of the protein on the regulation of the cell cycle. The study group comprises 27 indisputable cases of invasive lobular carcinoma showing varying degrees of cytological atypia. Immunohistochemical staining using well-characterized monoclonal and polyclonal antibodies disclosed cyclin D1 protein in the majority of the invasive lobular carcinoma cells in 80% of the tumors. In marked contrast, only rare cells of the noninvasive component (lobular carcinoma in situ) in the same tissue sections showed positive staining. Southern blotting of nine cases did not reveal evidence of cyclin D1 gene amplification. Immunohistochemical staining for Ki-67, a protein present in all dividing cells, showed that most cells positive for cyclin D1 did not stain for Ki-67. We conclude that the vast majority of invasive lobular carcinomas show overexpression of cyclin D1 protein. The absence of cyclin D1 protein expression in the noninvasive cells suggests that the molecule plays a role in the progression to the invasive form of lobular carcinoma. In contrast to the ductal types of breast cancer, cyclin D1 gene amplification does not seem to cause the cyclin D1 protein overexpression in lobular cancers. The lack of correlation between cyclin D1 and Ki-67 expression suggests that the cyclin D1 oncogene acts through mechanisms other than simple acceleration of the cell cycle clock in this subtype of human breast carcinoma.