Neurofilaments, 10 nm in diameter, from the axoplasm of the squid Loligo pealei have been isolated by a combination of sonication and Millipore filtration. The presence of neurofilaments during the isolation procedure was confirmed by negative staining and transmission electron microscopy. By use of this technique, which results in minimal or no chemical alteration of the native neurofilament proteins, it was shown that actin (43,000 daltons) and tubulin (56,000 daltons) are physically separable from intact neurofilaments. The neurofilament-rich retentate contained two major proteins of 200,000 and 63,000 daltons and larger polypeptides.