Complete nucleotide sequences have been derived for the Sabin type 3 vaccine, P3/Leon/l2a1b, and its neurovirulent progenitor, P3/Leon/37 (Stanway et al., 1983, 1984). These studies revealed 10 base substitution mutations which must account for the attenuated and temperature sensitive phenotypes of the vaccine. Complete DNA copies of the genomes of P3/Leon/12a1b and P3/Leon/37 were constructed, each within the prokaryote vector, pAT 153. The full-length cDNA clones were shown to be infectious following transfection of human epithelial cells. Virus rescued from the cDNA clone of P3/Leon/12a1b could not be distinguished from reference Preparations of the Sabin type 3 vaccine in standard assays for neurovirulence and temperature sensitivity. By the same criteria, virus rescued from the cDNA clone of P3/Leon/37 was shown to be identical to the parental strain. To determine the genetic basis for the attenuated phenotype, a series of inter-strain poliovirus recombinants were constructed via cDNA in-vitro. Attenuation results from the concerted effect of two independent point mutations. The first is a C-U substitution at position 472 in the 5' non-coding region of the viral genome. The second is a C-U substitution at position 2034 which results in a serine to phenylalanine substitution in VP3. The VP3 mutation confers a temperature sensitive phenotype. This appears to be the only temperature sensitive mutation in the vaccine.