Amino acid-defined diets deficient in methyl groups have been shown to result in a very high incidence of hepatocellular carcinoma. It has been suggested that this is a result of decreased levels of S-adenosylmethionine and the undermethylation of DNA. Accordingly, the enzyme glycine N-methyltransferase (GNMT, EC 220.127.116.11) may play a major role in maintaining the levels of S-adenosylmethionine in liver in response to changes in dietary methionine. The effect of methyl-deficient, amino acid-defined diets on GNMT activity and S-adenosylmethionine levels in rat liver was therefore investigated. When rats were fed a defined amino acid diet containing no choline in which homocysteine was substituted for the methionine of the control diet at an equimolar level, there was a rapid and marked decrease in growth rate in spite of the fact that the rats consumed 85% of the food eaten by control rats fed a nutritionally adequate, defined amino acid diet. The GNMT activity in livers of methyl-deficient rats decreased rapidly, but there was no difference in amount of GNMT protein as measured immunologically. In methyl-deficient rats, the levels of S-adenosylmethionine were maintained but the levels of S-adenosylhomocysteine were rapidly elevated compared to control values. These changes are consistent with the postulated role of GNMT in regulating methyl group metabolism.