The expression of the cyclin-dependent kinase inhibitor p15INK4B in normal cells after induction with TGF-beta1, or following overexpression from an adenovirus-encoded cDNA, appears on an SDS-polyacrylamide gel as a doublet. Here, the underlying mechanism behind the synthesis of the two species has been studied. By expressing cDNAs truncated at their 5' end, we found that the synthesis of the more slowly migrating form, called p15.5INK4B, is dependent on a sequence upstream of the first AUG codon thought to initiate translation of p15INK4B. Two potential, in frame, alternative upstream initiation codons, ACG and GUG, were individually changed to GCA encoding alanine. Analysis by in vitro translation, or immunoblotting of lysates from transfected 293 cells, showed that translation of p15.5INK4B is initiated at the GUG located 13 codons upstream of the first AUG. When this AUG was mutated, p15INK4B was no longer made. Instead, a shorter form, initiated at an in frame AUG located seven codons downstream, was synthesized. Finally, when both these AUGs were mutated, only p15.5INK4B was generated. Both p15INK4B and p15.5INK4B bound to CDK4 and CDK6, inhibited DNA synthesis, and caused replicative senescence of a human glioma cell line. We thus conclude that p15INK4B and p15.5INK4B, encoded by the CDKN2B gene, are functionally indistinguishable as based on these assays.