Proteins perform their activities in cells by the cooperation within protein complexes. For this reason, it is important to investigate protein-protein interactions to receive insights in physiological processes. A multitude of proteins are involved in the regulation of the cell cycle. Specific key factors participating here are members of the E2F transcription factors. Using an in vivo protein-protein complex detection assay, which comprises mass spectrometric and immunological techniques, we detected a number of known as well as new protein-protein interactions. We describe here for the first time protein complexes containing the corepressor Alien and members of the E2F transcription factor family. Furthermore, we assessed the functional relevance and show a repression of the transcriptional activity of E2F by Alien. Additionally, we detected new interactions that link endogenously expressed Alien with the tumor suppressor retinoblastoma protein (pRB) and with proteins involved in cell cycle regulation.