Hydroxyproline (Hyp) in decaying organic matter is a rich source of carbon and nitrogen for microorganisms. A bacterial pathway for Hyp catabolism is known; however, genes and function relationships are not established. In the pathway, trans-4-hydroxy-l-proline (4-l-Hyp) is epimerized to cis-4-hydroxy-d-proline (4-d-Hyp), and then, in three enzymatic reactions, the d-isomer is converted via Δ-pyrroline-4-hydroxy-2-carboxylate (HPC) and α-ketoglutarate semialdehyde (KGSA) to α-ketoglutarate (KG). Here a transcriptional analysis of cells growing on 4-l-Hyp, and the regulation and functions of genes from a Hyp catabolism locus of the legume endosymbiont Sinorhizobium meliloti are reported. Fourteen hydroxyproline catabolism genes (hyp), in five transcripts hypR, hypD, hypH, hypST and hypMNPQO(RE)XYZ, were negatively regulated by hypR. hypRE was shown to encode 4-hydroxyproline 2-epimerase and a hypRE mutant grew with 4-d-Hyp but not 4-l-Hyp. hypO, hypD and hypH are predicted to encode 4-d-Hyp oxidase, HPC deaminase and α-KGSA dehydrogenase respectively. The functions for hypS, hypT, hypX, hypY and hypZ remain to be determined. The data suggest 4-Hyp is converted to the tricarboxylic acid cycle intermediate α-ketoglutarate via the pathway established biochemically for Pseudomonas. This report describes the first molecular characterization of a Hyp catabolism locus.