The mRNA of the intronless, single-copy CCAAT/enhancer-binding protein-beta (C/EBPbeta) gene encodes several isoforms that have truncated transcription activation domains. This occurs by the alternative translational initiation (ATI) at multiple AUG start sites. The C/EBPbeta mRNA has four in-frame AUGs and an internal out-of-frame AUG associated with a small open reading frame (sORF). Initiation of translation at the in-frame AUGs forms 40-kDa (AUG-1), 35-kDa (AUG-2), 20-kDa (AUG-3) and 8.5-kDa (AUG-4) isoforms. We show that in COS-1 cells the 20-kDa isoform is not a product of proteolysis of the higher molecular weight isoforms. The sORF contains an AUG and termination signal that may produce the oligopeptide MPPAAARRL. Our studies suggest that ATI involves three mRNA structural features: (i) the cap structure, (ii) the context of the Kozak sequences that flank the AUG and (iii) the integrity of the sORF. We propose that formation of C/EBPbeta isoforms is accomplished by a leaky ribosomal scanning mechanism that facilitates ATI of multiple internal AUGs.