Abstract, We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo ll6-kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesininduced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP-sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility.V ARIOUS forms of intracellular transport are thought to depend upon microtubule-associated mechanochemical ATPases (Vale, 1987). One such enzyme, kinesin (Vale et al., 1985a), binds microtubules in a nucleotide sensitive fashion, and displays a microtubule-activated ATPase activity that drives movement of kinesin-coated objects towards the plus end of microtubules (MTs) ~ in vitro (Vale et al., 1985a, b;Scholey et al., 1985;Brady, 1985; Kuznetsoy and Gelfand, 1986;Porter et al., 1987;Cohn et al., 1987;Saxton et al., 1988;Gelles et al., 1988). Kinesin from a variety of animals contains a major polypeptide with an apparent molecular mass of 110-140 kD, encoded by a single gene in Drosophila (Yang et al., 1988), plus copurifying light chains of Mr = 40-80 kD (Vale et al., 1985a,b;Amos, 1987;Kuznetsov and Gelfand, 1986;Saxton et al., 1988). Bovine brain kinesin is an tt2132 heterotetramer consisting of two 120-kD heavy chains plus two 60-kD light chains, assembled into an elongated molecule (Amos, 1987;Kuznetsov et al., 1988;Bloom et al., 1988). Kinesin is proposed to participate in organelle/vesicle transport, in organizing the endomembrane system, and in mitosis (Vale et al., 1986;Vale, 1987;Leslie et al., 1987;Schroer and Sheetz, 1988), but direct evidence concerning the biological functions of kinesin is currently lacking.Antibodies that inhibit mechanochemical activity have been extremely useful in probing the functions of myosin and 1. Abbreviations used in this paper: MT(s), microtubule(s); PMEG, 0.9 M glycerol, 0.1 M Pipes, pH 6.9, 5 mM EGTA, 2.5 mM MgSO4, 0.5 mM EDTA, 1 mM DTT, 100 Ixg/ml soybean trypsin inhibitor, 1 mg/mlp-tosyl-Larginine methyl ester hydrochloride, 10 Ixg/ml leupeptin, pepstatin, and aprotinin; RT, room temperature. dynein. For example, an antiserum that inhibits the ATPase activity of sea urchin sperm dynein caused a corresponding decrease in the beat frequency of reactivated sea urchin sperm, supporting the hypothesis that the dynein ATPase drives axonemal motility (Ogawa and Mohri, 1975;O...