Synergistes jonesii is a rumen bacterium that degrades toxic pyridinediols from Leucaena leucocephala. This work presents progress on the characterization of the degradation of dihydroxypyridines from L. leucocephala by S. jonesii, and particularly by its cell free extracts. The substrate 3-hydroxy-4-[1H]-pyridone (3,4-DHP) induced degradation of both 3,4- and 2,3-dihydroxypyridine (2,3-DHP) isomers, while 2,3-DHP induced only degradation of 2,3-DHP. The 2,3-DHP was an intermediate of the degradation of 3,4-DHP by cultures. The compound 2,6-dihydroxypyridine inhibited degradation of 2,3-DHP by extracts of cells previously induced with this substrate. Cell-free extracts from S. jonesii had a high hydrogenase activity and degraded 2,3-DHP anaerobically either in the presence of methyl viologen under H2 or in the presence of α-ketoacids under H2 or N2. Specific activity of 2,3-DHP degradation was increased when FAD+ or CoA were present. The pyridine ring of the 2,3-DHP is enzymatically reduced by S. jonesii in reactions that demand reducing power provided by hydrogenase activity or by the metabolism of pyruvate. An unidentified non-polar amino compound appeared in thin layer chromatography as 2,3-DHP was degraded. We anticipate that gas chromatography mass spectrometry will permit the purification and identification of products from the cell-free reaction mix, which represents a simpler system than cell cultures, so that a pathway for pyridinediol metabolism by S. jonesii can be proposed.