The directionality of the actin-dependent motors on squid axoplasmic organelles was determined using actin filaments assembled on the barbed ends of acrosomal processes. Acrosomal processes were isolated from Limulus polyphemus sperm and incubated in monomeric actin under conditions that promoted barbed end assembly only. Newly assembled actin was stabilized and stained with rhodamine-phalloidin and the presence of filaments at the barbed ends of the acrosomal processes was verified by fluorescence microscopy and negative contrast electron microscopy. Axoplasmic organelles that dissociated from extruded axoplasm were observed by video microscopy to move along the newly assembled actin filaments at an average velocity of 1.1 +/- 0.3 microns/second. All organelles moved in the direction away from the acrosomal fragment and towards the tip of the actin filaments. Therefore, the actin-dependent organelle motor on axoplasmic organelles is a barbed-end-directed motor like other myosins analyzed. These findings support the conclusions that axoplasmic organelles are driven by a myosin-like motor along actin filaments and that these filaments as well as microtubules function in fast axonal transport.