The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons). To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation. The N‐terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in‐phase region 471 bp downstream. A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids. The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha‐beta‐galactosidase and IF2 beta‐beta‐galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus. A deletion of the 5′‐non‐translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta‐beta‐galactosidase but not IF2 alpha‐beta‐galactosidase. This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha. Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay. A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet‐tRNA and various labelled aminoacyl‐tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)