In vitro assembly of translation initiation complexes from higher eukaryotes requires purification of ribosomal subunits, eukaryotic initiation factors, and initiator tRNA from natural sources, and therefore yields only limited material for functional and structural studies. Here we describe a robust, affinity chromatography-based purification of eukaryotic 48S initiation complexes from rabbit reticulocyte lysate (RRL), which significantly reduces the number of individual purification steps. Hybrid RNA molecules, consisting of either a canonical 5' UTR or an internal ribosome entry site (IRES) RNA followed by a short open reading frame and a streptomycin aptamer sequence, are incubated in RRL to form 48S complexes. The assembly reaction is then applied to a dihydrostreptomycin-sepharose column; bound complexes are washed and specifically eluted upon addition of streptomycin. The eluted fractions are further purified by centrifugation through a sucrose density gradient to yield pure 48S particles. Using this purification scheme, properly assembled IRES-mediated as well as canonical 48S complexes were purified in milligram quantities.