We studied protein turnover in the gastrointestinal tract of adult fasted rats, since the mechanisms responsible for protein wasting in these tissues are poorly understood. Protein mass of stomach, small intestine, and colon decreased by 14-29 and 21-49% after 1 and 5 days of fasting, respectively. The fractional rate of in vivo protein synthesis (ks) was approximately 34% lower in the stomach after 1 and 5 days of fasting due to decreased capacity for protein synthesis (Cs). In small intestine and colon, ks was not different after 1 day, but was approximately 26% lower on day 5, mainly because of a reduction in Cs. Thus protein wasting in the stomach is primarily mediated by decreased protein synthesis but not in small intestine and colon during short-term fasting. To determine which proteolytic systems may be activated in the gut, we measured mRNA levels for critical components of the lysosomal (cathepsins B and D), Ca(2+)-activated (m-calpain), and ubiquitin-dependent (ubiquitin, 14-kDa ubiquitin-conjugating enzyme E2, and C8, and C9 proteasome subunits) proteolytic pathways. mRNA levels for most of these components increased during fasting, suggesting that a coordinated activation of multiple proteolytic systems contributed to intestinal protein wasting.