The subcellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using 13 C NMR, accumulation of ␤-hydroxypropionate from [2-13 C]propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants, including both monocots and dicots. Examination of coding sequences and subcellular targeting elements indicated that the completed genome of A. thaliana likely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, Arabidopsis mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to ␤-hydroxypropionate by a modified ␤-oxidation pathway. The chy1-3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of ␤-hydroxyisobutyrate from exogenous isobutyrate, but not the accumulation of ␤-hydroxypropionate from exogenous propionate. The chy1-3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine.13 C NMR analysis of Arabidopsis seedlings exposed to [U-13 C]valine did not show an accumulation of ␤-hydroxypropionate. No evidence was observed for a modified ␤-oxidation of valine.13 C NMR analysis showed that valine was converted to leucine through the production of ␣-ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified ␤-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine.Propionate, in the form of propionyl-CoA, is produced from a number of metabolic precursors in higher eukaryotes. It is the final product of odd-chain fatty acid ␤-oxidation (1). It is also produced during the catabolism of several amino acids, including isoleucine, methionine, and valine (1, 2). Propionyl-CoA is also a final product of metabolism of the branched acid, phytanic acid, derived from the degradation of chlorophyll (3). Aside from a basic understanding of metabolic biochemistry, the anabolic and catabolic pathways for propionyl-CoA are also of considerable importance in metabolic engineering of polyhydroxyalkanoates in plants, especially in the production of mixed polyhydroxyalkanoate polymers that have relied on the use of propionyl-CoA as a metabolic intermediate (4, 5). Several pathways have been confirmed for the catabolism of propionylCoA (6 -8). Bacteria and yeast utilize a 2-methylcitrate pathway with reactions analogous to those of the tricarboxylic acid cycle and glyoxylate cycle (6). Mammals use a well established b...