Two major pepsinogens, PG1 and PG2, and one minor pepsinogen, PG3, were purified from the gastric mucosa of African coelacanth, Latimeria chalumnae (Actinistia). PG1 and PG2 were much less acidic than PG3. Their molecular masses were estimated by SDS-PAGE to be 37.0, 37.0 and 39.3 kD, respectively. When incubated at pH 2.0, PG1 and PG2 were converted autocatalytically to the mature pepsins through an intermediate form, whereas PG3 was converted to an intermediate form, but not to the mature pepsin autocatalytically. The N-terminal sequencing indicated that the 42 residue sequences of the propeptides of PG1 and PG2 were essentially identical with each other, but different from that of PG3. A phylogenetic tree based on the N-terminal propeptide sequences indicates that PG1 and PG2 belong to the pepsinogen A group, and PG3 to the pepsinogen C group. From the phylogenetic comparison, coelacanth PG1 and PG2 appear to be evolutionally closer to tetrapod pepsinogens A than ray-finned fish pepsinogens A, consistent with the traditional systematics. Pepsins 1 and 2 were essentially identical with each other and rather similar to mammalian pepsins A in the pH optimum toward hemoglobin (pH 2-2.5), the cleavage specificity toward oxidized insulin B chain and strong inhibition by pepstatin, except that they possessed a significant level of activity in the higher pH range unlike mammalian pepsins A.