Rhizobium meliloti is known to use betaines synthesized by its host, Medicago sativa, as osmoprotectants and sources of energy. It is shown in the present report that the symbiotic megaplasmid (pSym) of R. meliloti RCR2011 encodes functions essential to the catabolism of three betaines, trigonelline (nicotinic acid N-methylbetaine), stachydrine (proline betaine or dimethylproline), and carnitine (gamma-trimethyl-beta-hydroxybutyrobetaine). Preliminary evidence is presented showing that functions on pSym also influence the catabolism of choline and its oxidative product, glycine betaine. Genes implicated in betaine catabolism are found in the symbiotic region of pSym. Trigonelline catabolism functions lie between two clusters of symbiotic genes, nifKDH and nok/fixVI'. Stachydrine and carnitine functions lie to the right of trigonelline catabolism functions, immediately to the right of fixVI'. Information necessary to choline and glycine betaine catabolism is probably encoded to the right of stachydrine catabolism functions.