We have established conditions for the fractionation of cytoplasmic and membrane-bound polyribosomes from the clonal mouse cell line BC3H-1. Polyribosome fractions are obtained in good yield and purity. They are active in protein synthesis when incubated with nuclease-treated rabbit reticulocyte lysates, and we have demonstrated that the cytoplasmic and membrane-bound fractions direct the synthesis of distinctly different sets of proteins. Using immunoprecipitation and sodium dodecyl sulfate gel analysis, we have shown that the membrane-bound but not the cytoplasmic polyribosomes direct the synthesis of two protein species (Mr = 39,000 nd 42,000) which are homologous to the native alpha subunit of acetylcholine receptor. Peptide maps suggest that the two species synthesized in vitro may correspond to the nonglycosylated and glycosylated forms, respectively, of the alpha subunit.