Neurofilament number and subunit composition, which are highly regulated during development, have been proposed to help regulate axonal diameter and stability. From experiments on dissociated cell cultures of Xenopus laevis embryonic spinal cord, we have obtained direct evidence that neurofilaments help maintain the structural integrity of newly developing axons. An anti-neurofilament monoclonal antibody specific for Xenopus NF-M and the cell lineage tracer, lysinated FITC-dextran, were coinjected into a single blastomere of 2- cell stage embryos. Within neurons descended from the injected blastomere, this antibody specifically confined neurofilaments to the cell body for the first two days of culture, as assayed by immunocytochemical staining with antiserum against the low molecular weight neurofilament protein XNIF. Although whole IgGs and Fab fragments both affected neurofilament distribution, the whole IgGs were more effective. For the first 9 hr of culture, neurites containing anti- NF-M developed normally. By 21 hr, they were shorter than those of sibling control neurons within the same dish, and many became morphologically abnormal. Defects included large variations in diameter, poorly defined separations between the growth cone and neurite, and more collateral branching. Despite these abnormal features, neurons containing anti-NF-M had normal distributions of alpha-tubulin immunoreactivity and phalloidin-stained F-actin. These latter observations argued that defects resulted from the absence of neurofilaments rather than from interference of the movement of other structural materials essential for axonal growth. These results support the hypothesis that neurons use neurofilaments to help maintain the characteristic shapes of axons against the increasing structural demands placed upon the elongating process.