Cyclins have been demonstrated to mediate phosphorylation of the retinoblastoma tumor suppressor gene product (Rb) and/or to bind directly to Rb. Since Rb is a regulator of E2F and Sp1-mediated transcription, we have examined the effect of overexpression of cyclins on transcription mediated by E2F-dependent adenovirus E2 promoter and by Sp1 in a cotransfection assay in 3T3 cells. All the G1 cyclins tested, C, D1, D2, D3 and E, as well as cyclin A were able to stimulate E2 promoter activity to various levels with D3 showing the strongest stimulation. For stimulation of the E2 promoter by cyclins A, E and D-type cyclins was dependent upon the presence of functional E2F and ATF binding sites. Cyclin C, however, was able to stimulate both E2F and ATF-dependent transcription to the same level as the wild type E2 promoter. In addition, cyclin C was able to stimulate transcription mediated by Sp1, GAL4-Sp1 and GAL4-VP16, suggesting that cyclin C affects a general pathway of transcriptional activation. In contrast, cyclin D1 was able to repress specifically Sp1-mediated transcription through an Rb-independent pathway. These results suggest that cyclins can regulate transcription mediated by specific transcription factors in both positive and negative manners. Furthermore, the results demonstrate clear functional differences between the G1 cyclins, in particular, functional differences between the related D-type cyclins.