The pancreas efficiently absorbs amino acids for the synthesis of enzymes, but also secrets free amino acids in the pancreatic juice which are re-absorbed by the small intestine. Under free protein diet, the release of amino acids on the pancreatic juice (PJ) may play important role on the homeostasis maintenance of the small intestine. From the 20 proteinogenic amino acids analyzed in the PJ, L-glutamate (Glu) was 4 fold more concentrated in PJ when compared to plasma levels. The concentrative mechanism could be due to an increase in the transport of Glu and the synthesis of Glu in the exocrine pancreas. Since there is a high expression of neutral amino acids and L-glutamine (Gln) transporters in the pancreas and they localize to the basolateral membrane of the acinar cells, we hypothesize Glu is synthesized in acinar cells and secreted to the pancreatic juice. We analyzed at mRNA level the expression of the 2 glutaminase isoforms (gls1 and gls2), glutamine synthase (Glul), alanine aminotransferase 1 and 2 (GPT1, GPT2) and aspartate aminotransferase 1 and 2 (GOT1 and GOT2) in pancreas. Our results showed that the two glutaminase isoforms (Gls1 and Gls2) are expressed in the pancreas, GLS2 is localized in the acinar cells, and that the levels of gls2 and of the cytoplasmatic GPT were elevated in animals receiving diet free of protein. These results suggest Glu may be synthesized in the exocrine pancreas from Gln and L-alanine and that the dietary protein content can modulate the expression of enzymes involved in the synthesis of Glu. The secretory mechanism of Glu to PJ could involve zymogen vesicles and transport via apical membrane. Our experiments showed that Glu and its precursors were not concentrating in zymogen granules vesicles (ZGV) but were found at high concentrations in the cytoplasmic fraction, suggesting the secretory mechanism does not involve exocytosis. Immunofluorescence and western blotting of isolated ZGV suggested that the sodium-dependent Glu transporter EAAT1 (Slc1a3) is present on the zymogen vesicles membrane. Additionally, immunofluorescence of pancreatic tissue showed that EAAT1 localizes proximal to the apical membrane of acinar cells. Our results support the hypothesis that EAAT1 is involved in the secretion of Glu during vesicular docking to the apical membrane of acinar cells. These preliminary exciting results suggest a new mechanism for Glu concentration and secretion on the pancreatic juice, as well a recycling of neutral amino acids (L-alanine, Gln) and Glu between the pancreas and intestine.