Gastrin component I is the largest hormonally active form of gastrin. In order to determine its structure, we isolated progastrin-derived peptides from normal human antral tissue. A radioimmunoassay specific for sequence 20–25 of human progastrin was developed to monitor the purifications. After four or five steps of reverse-phase chromatography, the peptides were pure and could be identified by combination of microsequence, amino acid and mass spectral analysis as well as by a library of sequence-specific immunoassays. In addition to intact progastrin 1–80, fragments 1–71, 1–35, 6–35, 20–35, and 20–36 of progastrin were identified. Only the 71-amino-acid peptide contained at its C-terminus the α-amidated bioactive site (Trp-Met-Asp-Phe-NH2). This unoheptacontapeptide amide (gastrin-71) corresponds to component I and is the largest possible bioactive product of progastrin. Its structure shows that progastrin is used in its entirety for biosynthesis of active peptides. The occurrence of fragments 6–35, 20–35, and 20–36 demonstrate that antral progastrin is partially cleaved at two monobasic sites (Arg5 and Arg19) in addition to processing at the three C-terminal dibasic sites. The results show that both the N- and C-terminal parts of antral progastrin undergo extensive processing. The results also suggest that progastrin may follow two different processing pathways of which the less trafficked releases gastrin-71.