The influence of different physical forms of substrates on fibroblast behavior was examined by comparing the following experimental and control groups: 1) glass coverslips, 2) poly(methyl methacrylate-co-ethyl acrylate) (PMMAEA) cast films, 3) electrospun PMMAEA nanofibers, 4) electrospun PMMAEA/collagen nanofibers, and 5) electrospun collagen. Cell adhesion, spreading and proliferation were compared on the different substrates. It was observed that fibroblasts on electrospun PMMAEA, PMMAEA-collagen, and collagen substrates spread more slowly after plating, and did not spread out to the extent observed for glass or PMMAEA films. Cells on electrospun fibers exhibited more filopodial-like structures and fewer stress fibers than the glass and PMMAEA film surfaces. Cell viability studies showed that although cells remained viable on all substrates, proliferation was faster on glass and PMMAEA films than on electrospun substrates. Overall, fibroblast behavior appeared to more closely resemble in vivo behavior on the electrospun nanofibers than on films or glass substrates.