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The early development of the digestive biochemistry of three cultured Atherinopsids (two Odontesthes species and Chirostoma estor) was investigated. Relatively high lipolytic and high alkaline proteolytic activities were found in all species. In addition, gene expression of lipase and trypsin during the early development of C. estor not only indicates an early onset of the lipase activity but also demonstrates a major importance of protein digestion in this species. However, despite all of them share similar habitats, differences in their digestive activities were evident. The rise in the activities of brush border membrane enzymes correlated with the decrease in cytosolic activities, used as indicator of maturation of the digestive tract, was detected in both species of Odontesthes at 9 weeks after hatching. In contrast, no similar signs were measured in C. estor, this being in agreement to the long weaning period (nearly 5 months) reported for this species.