To shed light on how axonal transport is regulated, we examined the possible roles of protein kinase A (PKA) in vivo suggested by our previous work (Sato-Yoshitake et al., 1992). Pharmacological probes or the purified catalytic subunit of PKA were applied to the permeabilized- reactivated model of crayfish walking leg giant axon, and the effect was monitored by the quantitative video-enhanced light microscopy and the quantitative electron microscopy. Dibutyryl cyclic AMP caused concentration-dependent transient reduction in the number of anterogradely transported small vesicles, while the retrogradely transported organelles and anterogradely transported mitochondria showed no decrease. This transient selective inhibition of anterograde vesicle transport was reversed by the application of a specific inhibitor of PKA (KT5720) in a concentration-dependent manner, and was reproduced by the application of the purified catalytic subunit of PKA and augmented by the application of adenosine 5′-O-(3-thiotriphosphate) (ATP gamma S). Corresponding biochemical study showed that several axoplasmic proteins including kinesin were specifically phosphorylated by the activation of the PKA pathway. These findings suggest the possible roles of PKA in the regulation of the direction of the axonal transport in vivo. The finding that only vesicle transport but not mitochondria transport was inhibited also suggests that the transport of vesicles and that of mitochondria are differently regulated and might be supported by different motors.