Although amino acids (AA) synthesized by enteric microbiota in the upper gut of nonruminants can be absorbed, they do not necessarily make a net contribution to the host's AA supply. That depends on whether protein or nonprotein nitrogen sources are used for microbial protein production. We determined the contributions of urea, endogenous protein (EP), and dietary protein (DP) to microbial valine (M.VAL) at the distal ileum of growing pigs, based on isotope dilutions after a 4-d continuous infusion of L-[1-13C]valine to label EP and of [15N15N]urea. Eight barrows were assigned to either a cornstarch and soybean meal-based diet with or without 12% added fermentable fiber from pectin. Dietary pectin did not affect (P >0.10) the contributions of the endogenous and DP to M.VAL. More than 92% of valine in microbial protein in the upper gut was derived from preformed AA from endogenous and DP, suggesting that de novo synthesis makes only a small contribution to microbial AA. J. Nutr. 139: 1088–1094, 2009.