Ribosomes in all organisms contain oligomeric and flexible proteins called stalks, which are responsible for the recruitment of translational GTPase factors to the ribosome. Archaeal ribosomes have three stalk homodimers (aP1)(2) that constitute a heptameric complex with the anchor protein aP0. We investigated the factor binding ability of aP1 proteins assembled onto aP0, by gel-retardation assays. The isolated aP0(aP1)(2)(aP1)(2)(aP1)(2) complex, as well as the form bound to the Escherichia coli 50S core, as a hybrid 50S particle, interacted strongly with elongation factor aEF2, but weakly with aEF1A. These interactions were disrupted by a point mutation, F107S, at the C-terminus of aP1. To examine the ability of each copy of aP0-associated aP1 to bind to elongation factors, we constructed aP0.aP1 variant trimers, composed of an aP0 mutant and a single (aP1) 2 dimer. Biochemical and quantitative analyses revealed that the resultant three trimers, aP0(aP1)(2)(I), aP0(aP1)(2)(II), and aP0(aP1)(2)(III), individually bound two molecules of aEF2, suggesting that each copy of the aP1 C-terminal region in the aP0.aP1 trimers can bind tightly to aEF2. Interestingly, the unstable binding of aEF1A to each of the three aP0.aP1 trimers was remarkably stabilized in the presence of aEF2. The stability of the aEF1A binding to the stalk complex may be affected by the presence of aEF2 bound to the complex, by an unknown mechanism. (C) 2016 Elsevier Inc. All rights reserved.