Bacterial translation initiation factor IF1 is homologous to archaeal aIF1A and eukaryal eIF1A, which form a complex with their homologous IF2-like factors (aIF5B and eIF5B respectively) during initiation of protein synthesis. A similar IF1–IF2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30S–IF2 interaction by IF1. Here we compare Escherichia coli IF1 with thermophilic factors from Bacillus stearothermophilus and Thermus thermophilus. All three IF1s are structurally similar and functionally interchangeable in vivo and in vitro. However, the thermophilic factors do not stimulate ribosomal binding of IF2ΔN, regardless of 30S subunits and IF2 origin. We conclude that an IF1–IF2 interaction is not universally conserved and is not essential for cell survival.