The objective of this experiment was to determine the capability of ruminant gut tissues to detoxify ammonia-N using short-term incubations of isolated cells in vitro. Ruminal epithelial cells (REC) and duodenal mucosal cells (DMC) were isolated from growing Texel-Polypay ram lambs (n = 4) fed a pelleted forage:concentrate-based diet. Immediately after isolation, primary cells were incubated for 60 min with glucose (1mM), glutamate (1mM), [15N]ammonium chloride (5, 10, 20, or 40 mM), and 1 of 4 combinations of substrates (1 mM each) that could support urea synthesis [control, N-carbamoylglutamate (NCG); NCG + ornithine (ONCG); and ONCG + aspartate (AONCG)]. Treatments were arranged in a 4 x 4 factorial design. Incorporation of ammonia-15N into alanine, citrulline, arginine, and urea was determined by gas chromatography-mass spectrometry. For both cell types, ammonia-N transfer to alanine was lower when incubation medium contained NCG compared with control, whereas use of ammonia-N for net alanine synthesis increased quadratically with ammonia concentration regardless of substrate treatment. For REC, ammonia-N was not incorporated into citrulline, arginine, or urea, nor into arginine or urea by DMC. Ammonia-N use for net citrulline synthesis exhibited an inverse relationship with ammonia concentration, decreasing linearly as media ammonia concentration increased. Thus, ala-nine synthesis may be a significant metabolic pathway for ruminant gut tissues to detoxify ammonia-N when it is presented luminally at high concentrations as compared with detoxification by the ornithine-urea cycle. Furthermore, DMC do exhibit a metabolic capability to incorporate ammonia-N into citrulline, but low or absent activity of downstream enzymes of the ornithine-urea cycle appears to limit ammonia-N transfers to urea.