A novel two-step,process was,developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1 M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electro phoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate, (PPi), and, excess, deoxyribonucleotides under alkali conditions. The results show that the 3-D polyqcrylamide gel network has a high immobilization capacity and the modified-PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10 mu L of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the ciude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be-incorporated into a DNA-chip format for high-throughput pyrosequencing analysis.