Recombinant Semliki Forest virus (rSFV), as a new kind of neurotropic vector system, has great potential of gene therapy for stroke. However, very little is known about its transduction characteristics in cerebral cortex or corpus callosum (CC) in vivo, which are common targets for gene transfer in experimental stroke therapy. Here, we investigate and compare rSFV-mediated gene expression at above two brain regions in rat; 2.0 x 10(7) IU of rSFV encoding green fluorescent protein (rSFV-GFP) was locally injected into CC or cerebral cortex in two groups. At 36 h following injection, the number of GFP-positive cells, GFP distribution volume, and GFP expression level were examined in the rat brain of each group using continuous frozen sections and enzyme-linked immunosorbent assay. rSFV vector displayed noticeably different transduction patterns in CC and cerebral cortex in vivo. CC injection of vector increased GFP-positive cell number by 802%, GFP transduction volume by 958%, and GFP expression level by 508% compared with cortical injection (all P < 0.01). We concluded that rSFV CC delivery significantly enhances transduction efficiency in rat brain with its ability to achieve transgene extensive transduction and abundant expression, and CC may be a favorable target for improving rSFV-based gene delivery efficiency to brain.