Methionine dependence is a metabolic defect found thus far only in transformed and malignant cells. The defect is manifested as the inability of cells to grow in media in which methionine (Met) is replaced by its immediate precursor homocysteine (Hcy). We have termed this Met- Hcy+ media. We demonstrate here that methionine-dependent cells derived from human tumors, compared to normal methionine-independent cells, have low levels of free Met, low levels of S-adenosylmethionine (AdoMet) and elevated levels of S-adenosylhomocysteine (AdoHcy) when incubated in Met- Hcy+ medium. Methionine-independent human tumor cells also have very low levels of free Met compared to normal cells but generally have levels of AdoMet and AdoHcy comparable to normal cells in Met- Hcy+ medium. All tumor cell types incorporate amounts of Met into protein similar to normal methionine-independent human fibroblasts when incubated in Met- Hcy+ medium, thereby indicating apparently normal levels of Met synthesis in the tumor cells. The methionine-independent tumor cell lines in Met- Hcy+ medium seem able to regulate their AdoMet/AdoHcy ratios normally despite this defect in having very low levels of free Met. Thus, in a diverse set of human tumor cell lines, all are defective in at least one aspect of Met metabolism, giving rise to the possibility of a general metabolic defect in cancer.