The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. Four out of five ribosomal P proteins of Trypanosoma cruzi, TcP0, TcP1α, TcP2α, and TcP2β had been previously characterized. Data mining of the T. cruzi genome data base allowed the identification of the fifth member of this protein group, a novel P1 protein, named P1β. To gain insight into the assembly of the stalk, a yeast two-hybrid based protein interaction map was generated. A parasite specific profile of interactions amongst the ribosomal P proteins of T. cruzi was evident. The TcP0 protein was able to interact with all both P1 and both P2 proteins. Moreover, the interactions between P2β with P1α as well as with P2α were detected, as well as the ability of TcP2β to homodimerize. A quantitative evaluation of the interactions established that the strongest interacting pair was TcP0-TcP1β. © 2005 Elsevier B.V. All rights reserved.