The N21I missense mutation in the cationic trypsinogen gene is the second most frequent mutation in hereditary pancreatitis (HP). In this article, we suggest that the N21I mutation most likely arose as a gene conversion event in which the functional anionic trypsinogen gene acted as the donor sequence. This hypothesis is supported by the unique presence of Ile at residue 21 of the anionic gene amongst the several highly homologous trypsinogen genes; a single unbroken tract of nucleotides of up to 113 bp flanking the I21 residue in the anionic trypsinogen gene; and the presence of a chi-like sequence in the 5' proximity and a palindromic sequence in the 3' vicinity of the N21I mutation. Furthermore, a multiple alignment of the partial amino acid sequence of vertebrate trypsins around residue 21 indicated that N21 and I21 may represent advantageously selected mutations of the two functional human trypsinogen genes in evolutionary history. These observations, which are complementary to the previous findings, provide further insights into the genetic mechanism and pathogenic role of the N21I mutation in HP.