The microtubule-dependent motor protein Eg5 is essential for the development and function of the mitotic spindle. Now it has become an anti-mitotic drug target in high throughput screening for anticancer dugs in vitro. Here is a protocol for cloning, expression and purification of a human Eg5 that codes for motor and linker domain in Escherichia coli BL21 (DE3) cells. The effects of temperature, pH, metal ions and DMSO on ATPase activity were investigated. A new compound CPUYL064 showed good inhibitory effect against Eg5 (IC(50) value, 100 nM). It inhibited the proliferation of human hepatocellular liver carcinoma cell line HepG2 in a dose- and time-dependent manner. CPUYL064 induced a clear G(2)/M phase arrest and caused the monastral spindle in HepG2 cells. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential and by detection of DNA fragmentation. These results indicate that CPUYL064 could be developed as a new, potent mitotic arrest compound.