Gelatin-based nanofibrous scaffolds with a mean fiber diameter of 300 nm were prepared with and without micrometer-sized polyethylene glycol (PEG) fibers that served as sacrificial templates. Upon fabrication of the scaffolds via electrospinning, the gelatin fibers were crosslinked with glutaraldehyde, and the PEG templates were removed using tert-butanol to yield nanofibrous scaffolds with pore diameters ranging from 10 to 100 mu m, as estimated with mercury intrusion porosimetry. Non-templated gelatin-based nanofibrous matrices had an average pore size of 1 mu m. Fibroblasts were seeded onto both types of the gelatin-based nanofibrous surfaces and cultured for 14 days. For comparative purposes, chitosan-based and polyurethane-based macroporous scaffolds with pore sizes of 100 and 170 mu m, respectively, were also included. The number of cells as a function of the depth into the scaffold was judged and quantitatively assessed using nuclei staining. Cell penetration up to a depth of 250 and 90 mu m was noted in gelatin scaffolds prepared with sacrificial templates and gelatin-only nanofibrous scaffolds. Noticeably, scaffold preparation protocol presented here allowed the structural integrity to be maintained even with high template content (95%) and can easily be extended toward other classes of electrospun polymer matrices for tissue engineering.