Two assays have been devised to demonstrate ATP-dependent migration of 40S ribosomal subunits on messenger RNA. The first is a two-step runoff assay. Reovirus mRNA was initially loaded with 40S subunits by incubation with wheat germ ribosomes in the presence of the antibiotic edeine. During the second phase of the incubation, in which further attachment of ribosomes was inhibited, the preformed complexes were shown to dissociate (presumably by runoff) only if ATP was included in the reaction. A more direct demonstration of ATP-dependent migration of 40S subunits was carried out using 3' end-labeled brome mosaic virus mRNA. In the presence of edeine and ATP, 40S ribosomal subunits were shown to advance all the way to the 3' end of the message, as shown by protection of the labeled 3'-proximal segment against nuclease digestion. Depletion of ATP by the addition of hexokinase prevented this migration. A variety of observations has raised the possibility that attachment of eucaryotic ribosomes to messenger RNA proceeds via a "scanning mechanism." The hypothesis is that a 40S subunit binds initially at or near the 5' terminus of the message and subsequently migrates toward the interior, stopping when it encounters the first AUG triplet. If migration of 40S subunits requires ATP, as the present studies suggest, the scanning mechanism predicts that in a system depleted of ATP a single 40S ribosome should be trapped near the 5' terminus of the message--upstream of the AUG initiator codon. This prediction was confirmed by analyzing binding of wheat germ ribosomes to a synthetic ribopolymer in which the 5'-proximal region (lacking AUG codons) and the AUG-containing segment near the 3' end of the molecule were differentially labeled.