Vimentin (Vm) is initially expressed by early neuronal precursors in situ and in culture. Vm is essential for neuritogenesis at least in culture and is gradually replaced by neurofilaments (NFs) because of down-regulation of Vm expression. This period is accompanied by a slowing of axonal elongation. We examined whether continued expression of Vm could foster continued axonal elongation. NB2a/d1 cells differentiated with dibutyryl cAMP were transfected with constructs expressing Vm or the middle-molecular-weight NF subunit (NF-M) each conjugated to green fluorescent protein (GFP). Axonal neurites of cells expressing GFP-Vm were 30% longer than those of nonexpressing cells, or cells expressing GFP-M, and exhibited a decrease in neurite caliber. Expression of GFP-M did not enhance axonal neurite length but significantly increased caliber. These findings provide further evidence of a role for Vm in axonal outgrowth. Culturing of nontransfected cells on laminin increased neurite length, but cells expressing GFP-Vm demonstrated an equivalent increase whether cultured on laminin or culture plastic. Axonal neurites of cells expressing GFP-Vm turned to avoid a nonfavorable substrate (nitrocellulose), but culturing of these cells on nitrocellulose did not impair axonal outgrowth. These latter findings indicate that the more robust outgrowth following reexpression of Vm is independent of a favorable or nonfavorable substrate but that axonal neurites of these cells still interact with the substrate to the extent that the substrate can influence directionality. © 2004 Wiley-Liss, Inc.