At some stage during initiation the ribosomal subunits of Escherichia coli must become irreversibly coupled, since polysomal ribosomes, in contrast to free ribosomes, are not dissociated by initiation factor IF-3. To determine when irreversibility develops we have compared the response to IF-3 of mature, puromycin-reactive initiation complexes, made with GTP, and of intermediate, puromycin-unreactive complexes, made with GMPPCP. The latter complexes initially appeared to be dissociated by the factor but this effect was found to be due to artificial loss of the ligands at the Mg2+ concentration customary in the test for dissociation. At a slightly higher Mg2+ concentration (4 mM), sufficient to retain the ligands, the GMPPCP complexes were not significantly dissociated by IF-3, at concentrations that caused complex dissociation of free ribosomes. It thus appears that the intermediate 70S initiation complex, though less stable to ionic dissociation than the mature complex, is in effect irreversible under physiological conditions.