Single microtubules from squid axoplasm support bidirectional movement of organelles. We previously purified a microtubule translocator (kinesin) that moves latex beads in only one direction along microtubules. In this study, a polar array of microtubules assembled off of centrosomes in vitro was used to demonstrate that kinesin moves latex beads from the minus to the plus ends of microtubules, a direction that corresponds to anterograde transport in the axon. A crude solubilized fraction from squid axoplasm (Sla), however, generates bidirectional movement of beads along microtubules. Retrograde bead movement (1.4 ymlsec) is inhibited by N-ethylmaleimide and 20 yM vanadate while anterograde movement (0.6, um/sec) is unaffected by these agents. Furthermore, a monoclonal antibody against kinesin, when coupled to Sepharose, removes the anterograde, but not the retrograde, bead translocator from Sla. These results indicate that there is a retrograde bead translocator which is pharmacologically and immunologically distinct from kinesin.