Methotrexate (MTX) affects homocysteine (Hcy) metabolism in both cultured cells and patients, and this may be explained by a lack of the 5-methyltetrahydrofolate required for salvage of Hcy to methionine. We here report the effect of MTX on Hcy in serum and Hcy, S-adenosylhomocysteine (AdoHcy), S-adenosylmethionine (AdoMet) and reduced glutathione (GSH) in tissues of rats fed either a normal or a defined, choline-deficient (CD) diet. The CD diet alone did not affect the amounts of Hcy in serum and tissues, but decreased the amount of AdoMet in most tissues and increased the GSH content in the liver. MTX increased the amount of Hcy about 2-fold in serum, liver and kidney, and decreased the amount of AdoMet in liver and kidney, whereas the AdoHcy content in these tissues was essentially unaffected. Accordingly, both choline deficiency and MTX treatment reduced the AdoMet to AdoHcy ratio. The increased GSH in the liver induced by CD diet seemed to be abolished by MTX. In the spleen MTX had only a marginal effect on the Hcy and AdoMet content and decreased the GSH content. It is concluded that the increase in serum Hcy during MTX exposure probably reflects a disturbance of the Hcy metabolism in some tissues, and especially in the liver. Altered metabolism of other sulfur-containing metabolites may only partly be related to the inhibition of Hcy salvage, and some metabolic effects of MTX may be modulated by tissue-specific metabolic pathways as well as by the diet.