The carboxy terminal S-farnesylcysteine of Ras oncoproteins is required for their membrane anchorage and transforming activities. We showed previously that S-farnesylthiosalicylic acid (FTS) affects the membrane anchorage of activated H-Ras in EJ cells and inhibits their growth. We report here on structural elements in S-prenyl derivatives that specifically inhibit the growth of EJ cells, but not of untransformed Rat-1 cells. Inhibition of the Ras-dependent extracellular signal-regulated protein kinase (ERK), of DNA synthesis and of EJ cell growth were apparent after treatment with FTS or its 5-fluoro, 5-chloro and 4-fluoro derivatives or with the C20 S-geranylgeranyl derivative of thiosalicylic acid. The 4-Cl-FTS analogue was a weak inhibitor of EJ cell growth. The 3-Cl-FTS analogue and the FTS carboxyl methyl ester were inactive, as were the C10 S-geranyl derivative of thiosalicylic acid, farnesoic acid, N-acetyl-S-farnesyl-L-cysteine and S-farne-sylthiopropionic acid. The structural requirements for anti-Ras activity of S-prenyl analogues thus appear to be rather stringent. With regard to chain length, the C15 farnesyl group linked to a rigid backbone seems to be necessary and sufficient. A free carboxyl group in an appropriately rigid orientation, as in thiosalicylic acid, is also required. Halogenic substitutents on the benzene ring of the thiosalicylic acid are tolerated only at position 5 or 4. This information may facilitate the design of potent Ras antagonists and deepen our understanding of the mode of association of Ras with the plasma membrane.