The retinoblastoma protein (RB) is a eukaryotic tumor suppressor that functions as a negative regulator of the cell cycle. In Chlamydomonas reinhardtii, a unicellular green alga, cells lacking the RB homolog MAT3 have defects in cell-size checkpoint control and cell cycle progression that lead to the production of tiny cells. We carried out a screen for suppressors of a mat3 null allele in order to dissect the cell cycle network that functions downstream of MAT3. The two strongest classes of suppressor had mutations in E2F1 and DP1--genes that encode subunits of a heterodimeric transcription factor that is regulated by retinoblastoma-related proteins in other species. We have subsequently used affinity purification and mass spectrometry to further characterize MAT3-containing protein complexes. Despite their extremely low abundance, both DP1 and E2F1 were identified as components of these complexes. We are now investigating additional proteins that appear to be associated with MAT3. The characterization of MAT3 protein complexes in a unicellular organism like Chlamydomonas should yield insights into how the RB pathway first evolved to regulate the cell cycle in ancestral eukaryotes.