Electrospun fibers have been fabricated for wide use as artificial tissue engineering scaffolds. In particular, fibers smaller than a cell body have been extensively employed to mimic natural extracellular matrix (ECM) and to explore specific responses by various cell types. We investigated the effects of various poly(lactic acid-co-glycolic acid) (PLGA) fiber features on embryonic hippocampal neurons in the early developmental stages in terms of initial axon formation (i.e., polarization) and axon orientation. We produced PLGA fibers that have average diameters ranging from 0.44 mu m to 2.2 mu m and different degrees of fiber alignment (16-58 degrees in angular standard deviation). After 22 h in culture, embryonic hippocampal neurons grown on PLGA fibers exhibited more axon formation with a 30-50% increase over those on spin-coated smooth PLGA films. This improvement was independent of fiber diameter and alignment; however, slightly more polarization was observed on the smaller fibers and the more aligned fibers. In addition, average axon length of the polarized embryonic hippocampal neurons was not significantly different among the PLGA fibers when compared with cells grown on spin-coated PLGA films. These findings suggest that fibers of subcellular diameters stimulate initial axon establishment and guide the direction of axonal extension; however, these fibers do not appear to affect the overall axon length. This information will be valuable in understanding the roles of subcellular features on neuron development and for the design of biomaterials for neural tissue interfacing. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 92A: 1.398-1406, 2010