The role of calmodulin (CaM) in organelle motility (fast axonal transport) in the axoplasm of the squid giant axon was evaluated directly using video-enhanced microscopy. Addition of 6 μM CaM to extruded squid axoplasm produced a 2.6-fold increase in the number of organelles moving per minute per unit area of axoplasm. When lower concentrations of CaM, including physiological concentration (2 μg/ml), were added to extruded axoplasm, the number of organelles moving was equally increased. CaM had no significant effect on the mean velocity of organelle translocations. The stimulatory effect of CaM was reduced significantly by the CaM inhibitors melittin (36 μM) and trifluoperazine (50 μM). Parvalbumin, a high-affinity calcium binding protein, did not stimulate motile activity. These results suggest that CaM is a positive regulator of fast axonal transport. At the molecular level, this regulation may involve microtubule-and/or actin-based motor proteins. Several possible molecular mechanisms are proposed.