Many different types of cancer cells have been shown to be methionine-dependent. These cells, unlike normal cells, grow poorly or not at all when methionine is replaced by its immediate precursor homocysteine in the growth medium (Met- Hcy+ medium). We have previously shown that apparently normal total amounts of methionine are synthesized by methionine-dependent SV40-transformed human fibroblasts. However, methionine-dependent cells in Met- Hcy+ medium accumulate reduced amounts of S-adenosylmethionine (AdoMet) and elevated amounts of S-adenosylhomocysteine (AdoHcy) that together probably limit growth. In this report, we demonstrate that the amount of free methionine is low in methionine-dependent SV40-transformed human fibroblasts in Met- Hcy+ medium compared to normal human diploid fibroblasts. In contrast, in Met+ Hcy- medium, the amount of free methionine is comparable in both cell types. The deficient pool of free methionine in methionine-dependent cells in Met- Hcy+ medium allows only low amounts of AdoMet to be formed. However, large amounts of the biosynthesized methionine are channeled into protein synthesis. Possible mechanisms are discussed to explain this cancer-associated metabolic defect.