In our studies on the molecular biology of human gastrin-releasing peptide (GRP), we have discovered an example of a change in translational reading frame apparently produced through alternative RNA splicing. Complementary DNAs prepared from a pulmonary carcinoid tumor rich in GRP immunoreactivity had one of two different-sized internal DNA fragments after digestion with the restriction enzyme Pvu II. Nucleotide sequences of the two DNA fragments were identical except for 19 additional nucleotides present in the larger fragment. The region of the mRNA containing the 19 nucleotides corresponded to the carboxyl-terminal region of the human GRP precursor. The resulting shift in reading frame causes a difference of 10 amino acids in size and an overall sequence difference of 27 amino acids between the two GRP prohormones so formed. The change in reading frame described here is unusual in eukaryotes and is yet another mechanism to produce diversity in the generation of biological peptides.