Since the cytocompatibility of carbon nanofibers with respect to neural applications remains largely uninvestigated, the objective of the present in vitro study was to determine cytocompatibility properties of formulations containing carbon nanofibers. Carbon fiber substrates were prepared from four different types of carbon fibers, two with nanoscale diameters (nanophase, or less than or equal to 100 nm) and two with conventional diameters (or greater than 200 nm). Within these two categories, both a high and a low surface energy fiber were investigated and tested. Astrocytes (glial scar tissue-forming cells) and pheochromocytoma cells (PC-12; neuronal-like cells) were seeded separately onto the substrates. Results provided the first evidence that astrocytes preferentially adhered on the carbon fiber that had the largest diameter and the lowest surface energy. PC-12 cells exhibited the most neurites on the carbon fiber with nanodimensions and low surface energy. These results may indicate that PC-12 cells prefer nanoscale carbon fibers while astrocytes prefer conventional scale fibers. A composite was formed from poly-carbonate urethane and the 60 nm carbon fiber. Composite substrates were thus formed using different weight percentages of this fiber in the polymer matrix. Increased astrocyte adherence and PC-12 neurite density corresponded to decreasing amounts of the carbon nanofibers in the poly-carbonate urethane matrices. Controlling carbon fiber diameter may be an approach for increasing implant contact with neurons and decreasing scar tissue formation.