Polysome analysis has proved to be a sensitive probe for the mode of action of inhibitors of protein synthesis in intact HeLa cells. To classify the active compounds as inhibitors of initiation, elongation, or termination, their effects on the cellular polyribosome pattern were compared under three conditions. These conditions tested (i) their direct effect on the polyribosome profile; (ii) their effect on ribosome run-off produced by hypertonicity; and (iii) their effects on recovery from hypertonicity. Using this technique, diacetoxyscirpenol, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and three alkaloids, harringtonine, isoharringtonine, and homoharringtonine, were found to be inhibitors of initiation. Polysome analysis indicated that in HeLa cells 7.8 x 10(-7) M pactamycin, which inhibited protein synthesis 94%, interfered with elongation as well as initiation under these conditions. Emetine, anisomycin, cycloheximide, and trichodermin each gave polysome patterns consistent with inhibition of elongation. Fusidic acid and aurintricarboxylic acid inhibited incorporation of [(14)C]leucine into intact HeLa cells, but polysome analysis did not localize any specific inhibitory effects to the initiation, elongation, or termination steps of protein synthesis. The use of specific inhibitors of initiation of protein synthesis has indicated that most, if not all, mammalian messenger ribonucleic acids contain a single initiation site.