Actin is one of the major cytoskeletal proteins carried in slow axonal transport. Since more than 50% of actin in the axon was recovered in the high-speed supernatant, we looked for G-actin-binding proteins in slow axonal transport. Two weeks after injection of L-[35S]methionine into the rat spinal cord (L3-L5), labeled proteins in the sciatic nerve were extracted and those with potential abilities to interact with G-actin were detected by two independent methods: (A) DNAase I affinity chromatography and (B) blot overlay with biotinylated actin. By method (A), a 68 kDa Ca(2+)-dependent binding protein and a 45 kDa Ca(2+)-independent binding protein were detected. The 68 kDa protein was also a major protein binding to actin in method (B). The 68 kDa protein was identified with the Ca(2+)-dependent phospholipid binding protein annexin VI by two-dimensional electrophoresis and Western blotting. As annexin VI is a component of slow axonal transport, it does not seem to be bound to membranous organelles in the axon. Our results suggest that annexin VI may play a role in the control of actin assembly and membrane-microfilament interaction.