Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N‐domain displayed neither affinity for ribosomes nor a detectable function, the isolated C‐domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S‐bound mRNA from ‘stand‐by’ to ‘P‐decoding’ site; (iii) dissociation of 30S–poly(U)–NacPhe‐tRNA pseudo‐ initiation complexes; (iv) dissociation of fMet‐tRNA from initiation complexes containing mRNA with the non‐canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C‐domain–30S interaction, the N‐domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S–IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem–loop of P‐site‐bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.