Overexpression of tau compromises axonal transport and induces retraction of growing neurites. We tested the hypothesis that increased stability provided by neurofilaments (NFs) may prevent axonal retraction. NB2a/d1 cells were differentiated for 3 days, at which time phosphorylated NFs appear and for 14 days, which induces continued neurite elongation and further phospho-NF accumulation. Cultures were transfected with a construct that expresses full-length, 4-repeat tau. Consistent with prior studies, overexpression of tau induced retraction of day three axonal neurites even following treatment with the microtubule-stabilizing drug taxol. Axonal neurites of day 14 cells were more resistant to tau-mediated retraction. To test whether or not this resistance was derived from their additional NF content, day 3 cultures were co-transfected with constructs expressing tau and NF-M (which increases overall axonal NFs). Overexpression of NF-M attenuated tau-mediated retraction of day 3 axonal neurites. By contrast, co-transfection with constructs expressing tau and vimentin (which increases axonal neurites length) did not attenuate tau-mediated neurite retraction. Co-precipitation experiments indicate that tau is a cargo of kinesin, and that tau overexpression may displace other kinesin-based cargo, including both critical cytoskeletal proteins and organelles. However, cultures simultaneously transfected with constructs expressing NF-M and tau, the level of examined vesicles was maintained. These collectively indicate that NFs stabilize developing axonal neurites and can counteract the destabilizing force resulting from overexpression of tau, and underscore that the development and stabilization of axonal neurites is dependent upon a balance of cytoskeletal elements. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc.