Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate glycosaminoglycans in the extracellular matrix and the basement membrane and is well known to be involved in tumor cell invasion and angiogenesis. We have focused on heparanase as a target for antitumor agents, especially antimetastatic agents. (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (RK-682) was found to display an inhibitory activity against heparanase in our screening of natural sources. Because RK-682 has been reported to show inhibitory activities against several enzymes, we have tried to develop selective heparanase inhibitors using the method of rational drug design. Based on the structure of the heparanase/RK-682 complex, we speculated that selective inhibitory activity against heparanase could be acquired by arylalkylation, namely, by benzylation of the 4-position of RK-682. Among the rationally designed 4-alkyl-RK-682 derivatives, 4-benzyl-RK-682 has been found to possess a selective inhibitory activity for heparanase (IC50 for heparanase, 17 micromol/L; IC50 for other enzymes, >100 micromol/L). 4-Benzyl-RK-682 also inhibited the invasion and migration of human fibrosarcoma HT1080 cells (IC50 for invasion, 1.5 micromol/L; IC50 for migration, 3.0 micromol/L). On the other hand, RK-682 had no inhibitory effect on the invasion and migration of HT1080 cells at doses of up to 100 micromol/L.