Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.