Human Eg5, responsible for the formation of the bipolar mitotic spindle, has been identified recently as one of the targets of S-trityl-L-cysteine, a potent tumor growth inhibitor in the NCI 60 tumor cell line screen. Here we show that in cell-based assays S-trityl-L-cysteine does not prevent cell cycle progression at the S or G 2 phases but inhibits both separation of the duplicated centrosomes and bipolar spindle formation, thereby blocking cells specifically in the M phase of the cell cycle with monoastral spindles. Following removal of S-trityl-L-cysteine, mitotically arrested cells exit mitosis normally. In vitro, S-trityl-L-cysteine targets the catalytic domain of Eg5 and inhibits Eg5 basal and microtubule-activated ATPase activity as well as mant-ADP release. S-Trityl-L-cysteine is a tight binding inhibitor (estimation of K i,app <150 nM at 300 mM NaCl and 600 nM at 25 mM KCl). S-Trityl-L-cysteine binds more tightly than monastrol because it has both an ϳ8-fold faster association rate and ϳ4-fold slower release rate (6.1 M ؊1 s ؊1 and 3.6 s ؊1 for S-trityl-L- Kinesins form a superfamily of motor proteins with about 14 different subfamilies clearly identified so far. They play important roles in intracellular transport and at different stages of cell division. The driving force behind these processes is ATP hydrolysis.The roles of different kinesins during cell division make them highly important for understanding fundamental aspects of mitosis and meiosis. In recent years, some of them have appeared as potential targets for anti-cancer drugs (1-3). One of these mitotic kinesins, human Eg5 (HsEg5/KSP), a member of the kinesin-5 family (4), is responsible for the formation and maintenance of the bipolar spindle (5). Eg5 represents an especially attractive target because when inhibited by microinjection with suitable antibodies (5), by RNAi 2 (6), or by treating cells with specific Eg5 inhibitors (7), it displays a very characteristic mitotic arrest phenotype, i.e. a monoastral spindle with an array of microtubules (MTs) emanating from a pair of nonseparated centrosomes surrounded by chromosomes. Cell-based as well as in vitro assays have led to the discovery of a series of inhibitors that target Eg5 and lead to mitotic arrest and cell death. Among these inhibitors are monastrol, the first Eg5 inhibitor discovered (7), terpendole E, identified from a fungal strain (8), HR22C16, structurally related to monastrol (9), CK0106023, a quinazolinone analogue representing the most potent Eg5 inhibitor identified so far (10), dihydropyrazoles (11), and S-trityl-L-cysteine (STLC) (12). Several of these inhibitors are currently intensively studied as potential anticancer drugs, as tools for studying fundamental processes in mitosis and function of its target (chemical genetics) (13), or simply as a model to understand the mechanisms of inhibition of this important class of proteins (14 -17).By using two small, preselected libraries from the NCI, we have recently identified several new inhibitors of human Eg5 activity, ...