The protein product of the retinoblastoma tumor suppressor gene (RB) has been demonstrated to bind c-Myc protein (Myc) in vitro. To determine whether RB regulates Myc transcriptional activity in vivo, GAL4-Myc chimeric expression plasmids were generated and cotransfected with a RB expression plasmid and a GAL4-dependent reporter plasmid. RB stimulated GAL4-Myc-mediated transcription, dependent upon a domain(s) in the amino-terminus of Myc. The stimulation of Myc-mediated transcription by RB was cell-type specific and was inhibited by SV40 T-antigen, but not by a T-antigen mutant defective in RB-binding. Moreover, RB mutants containing mutations in domain B of RB pocket were significantly reduced in their ability to stimulate GAL4-Myc mediated transcription. To determine whether RB and Myc interact in vivo either directly or indirectly, a two hybrid system was used where GAL4-Rb and Myc-VP16 expression constructs were cotransfected with a GAL4-dependent reporter plasmid. A significant increase of GAL4-dependent transcription was observed, dependent upon the presence of both GAL4-Rb and Myc-VP16 fusion proteins. Mutational analysis of the Myc-VP16 chimeric proteins suggests that the amino-terminus of Myc is essential for the interaction with RB. These results demonstrate that RB can regulate Myc-mediated transcription in vivo in a cell-type specific manner through protein-protein interactions.