Cryogenic electrospinning has previously been demonstrated for controlling the pore sizes of electrospun scaffolds, which has been impossible with traditional electrospinning processes. This article describes the application of the cryogenic technique to fabricate a bilayered electrospun poly(D,L-lactide) scaffold (BLES) in a single Uninterrupted process. The resulting BLES consisted of a traditional electrospun (ES) fibrous layer with a dense pore area of 17 +/- 3 mu m(2) adjacent to a cryogenic electrospun layer (CES) with a pore area of 3300 +/- 500 mu m(2). The significance of this bilayered scaffold was to mimic the anatomical structure of tissues with dense basement membrane followed by loose and highly porous connective tissue such as skin and blood vessels. Cell infiltration in the BLES was compared in vitro and in vivo. Both studies suggested the CES supported high cell infiltration, whereas the ES could serve as a physical barrier to prevent cell infiltration across the CES-ES boundary because of its size exclusion. The bilayered structure produced by this technique suggests a great potential for engineering tissues with similar architectures. (C) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 94A: 1141-1149, 2010.