Southern bluefin tuna (Thunnus maccoyii) ranching in South Australia is one of the most valuable finfish aquaculture industries in Australia. This industry is currently highly reliant upon the use of baitfish; however commercial pelleted diets for this species are now available. Despite the economic importance of these animals there is a general lack of knowledge regarding the digestive physiology of southern bluefin tuna. Measurement of the digestive enzymes trypsin (T), chymotrypsin (C) and calculation of T:C ratios are particularly useful in the assessment of nutritional status in fish. In this study T and C activities were measured in ranched tuna fed a vitamin (Vit) and vitamin+immunostimulant (Vit+Imm) supplemented baitfish diet for 8weeks. Differences relating to the addition of the supplements were not observed for T, C or T:C ratios suggesting that the supplementation rates did not affect the nutritional status of the tuna or the quality of the baitfish. Trypsin activity plays a key role in the secretion of chymotrypsin and in this study a significant correlation between these enzymes was found. T, C and T:C were not associated with caecum somatic index, caecum pH, caecum temperature, stomach pH or stomach temperature. Although T and C activities are highly sensitive to feeding, activities of these enzymes did not appear to be affected by the presence of baitfish in the stomach. The presence of baitfish did not appear to affect caecum pH, caecum temperature or stomach temperature. Therefore all data were pooled and a significant correlation between caecum and stomach temperature, explained by the endothermic physiology of tuna, was observed. Significantly higher stomach pH values were associated with the presence of baitfish and an inverse relationship between stomach pH and stomach temperature was found in fish with empty stomachs but not those that had eaten. These results are indicative of a digestive strategy which employs maintenance of low stomach pH for rapid digestion of a meal. This study represents the first report of digestive enzyme activities and nutritional status for captive southern bluefin tuna fed a standard and modified baitfish diet. In addition to a better understanding of digestive physiology, these results, in combination with the physiological measures of feeding and digestion, will significantly contribute to the further development of an in vitro digestive model for this species. This model will significantly improve the abilities of the Australian southern bluefin tuna industry to assess the predicted digestibility of commercially produced pelleted diets as well as screen novel dietary ingredients prior to full scale and often commercially expensive in vivo trials.