Conventional kinesin (Kinesin-1), the founding member of the kinesin family, was discovered in the squid giant axon, where it is thought to move organelles on microtubules. In this study, we identify a second squid kinesin by searching an expressed sequence tag database derived from the ganglia that give rise to the axon. The full-length open reading frame encodes a 1753 amino acid sequence that classifies this protein as a Kinesin-3. Immunoblots demonstrate that this kinesin, unlike Kinesin-1, is highly enriched in chaotropically stripped axoplasmic organelles, and immunogold electron microscopy (EM) demonstrates that Kinesin-3 is tightly bound to the surfaces of these organelles. Video microscopy shows that movements of purified organelles on microtubules are blocked, but organelles remain attached, in the presence Kinesin-3 antibody. Immunogold EM of axoplasmic spreads with antibody to Kinesin-3 decorates discrete sites on many, but not all, free organelles and localizes Kinesin-3 to organelle/microtubule interfaces. In contrast, label for Kinesin-1 decorates microtubules but not organelles. The presence of Kinesin-3 on purified organelles, the ability of an antibody to block their movements along microtubules, the tight association of Kinesin-3 with motile organelles and its distribution at the interface between native organelles and microtubules suggest that Kinesin-3 is a dominant motor in the axon for unidirectional movement of organelles along microtubules.