Amino acids are widely regarded as the most important sources of propionate in disorders of propionate metabolism. Propionate production was measured in the fasting state by continuous infusion of sodium [1-13C]propionate in three children with methylmalonic acidemia (MMA) and three with propionic acidemia (PA). The contribution of isoleucine, valine, threonine, and methionine catabolism to total propionate production was estimated by extrapolation from the hydroxylation of phenylalanine determined by a continuous-infusion [2H5]phenylalanine technique. The contribution of gut bacterial propionate production was determined by measuring total propionate production before and after treatment with oral metronidazole (10 to 20 mg/kg/d for 1 week). Amino acid catabolism accounted for a mean of 51.7% (range, 24.5% to 66.4%) of total propionate production. The mean decrease in propionate production after metronidazole was 22.2% +/- 8.5 (P less than .02); this percentage is likely to represent the minimum propionate production attributable to gut bacteria. Approximately 30% of total propionate production was unaccounted for, and is likely to arise primarily from odd-chain fatty acid catabolism in the fasting state. These results indicate that sources of propionate other than from protein catabolism are important in disorders of propionate metabolism, and explain the generally disappointing response to dietary protein restriction.