In previous studies of the ribosome cycle in mammalian protein synthesis we have shown that during that portion of the cycle in which the ribosomal subunits occur as free or native particles, that is, after they are released at chain termination and before they rejoin the polyribosomal messenger RNA, the 40 S subunits appear predominantly in two forms, of buoyant density 1.49 and 1.40 g per cm3, referred to respectively as 40 S h (for higher buoyant density) and 40 S l (for lower buoyant density) native subunits. The intraribosomal 40 S subunits (i.e. 40 S subunits coupled to 60 S subunits in monomeric ribosomes or polyribosomes) are of buoyant density 1.51 g per cm3. These differences in density reflect the fact that in addition to the structural proteins found in intraribosomal subunits, certain extra, exchangeable proteins are bound to the native 40 S subunits, about 90,000 daltons to the 40 S h form and 750,000 daltons to the 40 S l form. On the basis of this and other evidence we proposed a model of protein synthesis in which the 40 S ribosomal subunit, after release at chain termination, sequentially binds several proteins required for initiating another round of protein synthesis, becoming first the 40 S h and then the 40 S l native subunit. In this paper we report that aurintricarboxylic acid, at a concentration which inhibits initiation specifically, prevents the binding of the proteins which convert the 40 S h native subunit to the 40 S l form, so that runoff in the presence of aurintricarboxylic acid leads to the accumulation of 40 S h subunits and the disappearance of 40 S l subunits. Aurintricarboxylic acid also removes these proteins from pre-existing 40 S l subunits, converting them to particles identical in density with 40 S h subunits. A crude preparation of initiation factors, the ribosome KCl wash fraction, contains proteins which bind to ribosome-derived 40 S subunits (density 1.51 g per cm3), converting them to particles similar to 40 S h and 40 S l subunits (1.49 and 1.40 g per cm3). Aurintricarboxylic acid also prevents the binding of the KCl wash proteins which produce the 1.40 g per cm3 particles but not of those which yield the 1.49 g per cm3 particles. The binding of proteins to the subunit is 50% inhibited at a ratio of about 50 molecules of aurintricarboxylic acid per 40 S subunit, and the inhibition appears to be competitive. If the proteins binding to the 40 S l native subunit are indeed initiation factors, the inhibition of binding of these proteins by aurintricarboxylic acid explains the inhibition of initiation. The availability of an inhibitor acting at this site should prove useful.