Making use of the nucleotides sequence of the RNA genome (7,440 nt) of poliovirus, synthetic deoxyoligonucleotides, 60-70 nt in length are synthesized. The oligonucleotides that map to adjacent segments in the genome are designed such that they are of plus- and minus-strand polarity with the overlapping complementary sequences at their termini. The oligonucleotides are assembled by asymmetric PCR, and then, the segments are ligated directly into a plasmid. The segments are assembled stepwise via common unique restriction endonuclease cleavage sites to yield a full-length poliovirus complementary DNA (cDNA), carrying a phage T7 RNA polymerase promoter at the (left) 5' end. Genomic RNA is generated with a phage T7 RNA polymerase. The viral RNA is incubated in a cell-free extract, where it is translated and replicated, resulting in the de novo synthesis of poliovirus (PV). Finally, in vivo and in vitro experiments are carried out to confirm that infectious material isolated from the cell-free extract is indeed infectious PV.All components of the synthetic PV are generated by biochemical means. No virus-related structure or component that may have been generated previously in vivo is used as template or as building block for the viral particles. Our work shows that it is possible to synthesize an infectious agent in test tube by solely following instructions from a written sequence.