Microtubules have been demonstrated to be a substrate for organelle transport and particle translocation in vitro and in vivo. Subsequent to a previous report of inhibition of axonal transport of exogenous tracers in vivo using antiserum NS-20 against tubulin (Johnston et al: Brain Res. 1986), we now show disruption of particle movement in extruded squid axoplasm using this unique immunological probe. Using video-enhanced contract-differential interference contrast (AVEC-DIC) microscopy, we examined the properties of particle movement along microtubules and demonstrated that both the velocity of particle movement and the numbers of particles moving are decreased in the presence of NS-20 antiserum or NS-20 affinity-purified antibodies but not in the presence of another antiserum against tubulin. The amount of microtubule substrate does not change in the presence of any of the antisera. In conclusion, we suggest that NS-20 antibodies bind near or at a site on the tubulin molecule which is critical in the mechanism of particle transport, and provide a direct immunological probe to examine the mechanism of microtubule involvement in axonal transport.