A series of polyamine analogs has been examined for their ability to support protein synthesis in an in vitro rabbit reticulocyte translation system. Diamines were found to stimulate protein synthesis to the greatest extent (8–12 fold). Triamines, tetraamines and pentaamines only stimulated 2–4 fold under these conditions although much lower concentrations were required. At elevated temperatures (45°C), diamines were somewhat more active than at lower temperature but activity of longer chain polyamines was elevated very significantly. Polyamines with terminal benzyl or smaller alkyl groups had diminished activity. It is concluded that both charge and charge distribution determine the ability of polyamines to stimulate translation. Fidelity studies identified two classes of polyamines: those which are able to lower the optimal Mg 2+ concentration required for amino acid misincorporation while not affecting extent of misincorporation relative to Mg 2 alone, and those which are sparing for Mg 2+ and also stimulate extent of misincorporation.