The specific loss of pRB or p107 together with p130 disrupts the normal development of only a very limited spectrum of tissues. These developmental defects have been attributed primarily to deregulation of E2F activity and consequent uncontrolled proliferation. We hypothesized, however, that the tissue-specific nature of these defects may also reflect deregulation of pRB-family associated factors that are specifically involved in determining cell fate. We report here that the pRB-family members interact with transcription factors which contain paired-like homeodomains such as MHox, Chx10 and Pax-3. The interaction between the pRB-family and the paired-like homeodomain proteins was initially identified in a yeast two-hybrid screen where the N-terminal portion of p130 was used to isolate interacting factors from an embryonic mouse library. This interaction was confirmed by in vitro binding and co-immunoprecipitation assays. We show further that co-expression of Pax-3 dependent pRB, p107 or p130 with Pax-3 causes repression of activated transcription from the c-met promoter. These data demonstrate that the pRB-family proteins can modulate the activity of factors which specifically control cell fate and/or differentiation as well as controlling cell cycle regulators.