Betaine-homocysteine S-methyltransferase (BHMT) is the only enzyme known to catabolize betaine. In addition to being a substrate for BHMT, betaine also functions as an osmoprotectant that accumulates in the kidney medulla under conditions of high extracellular osmolarity. The mechanisms that regulate the partitioning of betaine between its use as a methyl donor and its accumulation as an osmoprotectant are not completely understood. The aim of this study was to determine if BHMT expression is regulated by salt intake. This report shows that guinea pigs express BHMT in the liver, kidney and pancreas, and that the steady-state levels of BHMT mRNA in kidney and liver decrease 68% and 93% in guinea pigs consuming tap water containing high levels of salt compared to animals provided untreated tap water. The animals consuming the salt water also had about 50% less BHMT activity in the liver and kidney, and steady-state protein levels decreased about 30% in both organs. Pancreatic BHMT activity and protein levels were unaffected by the high salt treatment. The complex mechanisms involved in the down-regulation of hepatic and renal BHMT expression in guinea pigs drinking salt water remain to be clarified, but the physiological significance of this downregulation may be to expedite the transport and accumulation of betaine into the kidney medulla under conditions of high extracellular osmolarity.