The retinoblastoma protein (pRb) functions as a regulator of cell proliferation and in turn is regulated by cyclin-dependent kinases. Cyclins D1 and D3 can form complexes with pRb that resemble those formed by several viral oncoproteins and are disrupted by the adenovirus E1A oncoprotein and derived peptides. These cyclins contain a sequence motif similar to the pRb-binding conserved region II motif of the viral oncoproteins. Alteration of this motif in cyclin D1 prevents formation of cyclin D1-pRb complexes while enhancing the biological activity of cyclin D1 assayed in vivo. We conclude that cyclins D1 and D3 interact with pRb in a fashion distinct from cyclins A and E, which can induce pRb hyperphosphorylation, and that cyclin D1 activity may be regulated by its association with pRb.