The complete nucleotide sequence of the RSV cpts-248/404 live attenuated vaccine candidate was determined from cloned cDNA and was compared to that of the RSV A2/HEK7 wild-type, cold-passaged cp-RSV, and cpts-248 virus, which constitute the series of progenitor viruses. RSV cpts-248/404 is more attenuated and more temperature sensitive (ts) (shut-off temperature 36 degrees) than its cpts-248 parent virus (shut-off temperature 38 degrees) and is currently being evaluated in phase I clinical trials in humans. Our ultimate goal is to identify the genetic basis for the host range attenuation phenotype exhibited by cp-RSV (i.e., efficient replication in tissue culture but decreased replication in chimpanzees and humans) and for the ts and attenuation phenotypes of its chemically mutagenized derivatives, cpts-248 and cpts-248/404. Compared with its cpts-248 parent, the cpts-248/404 virus possesses an amino acid change in the polymerase (L) protein and a single nucleotide substitution in the M2 gene start sequence. In total, the cpts-248/404 mutant differs from its wild-type RSV A2/HEK7 progenitor in seven amino acids [four in the polymerase (L) protein, two in the fusion (F) glycoprotein, and one in the (N) nucleoprotein] and one nucleotide difference in the M2 gene start sequence. Heterogeneity at nucleotide position 4 (G or C, negative sense, compared to G in the RSV A2/HEK7 progenitor) in the leader region of vRNA developed during passage of the cpts-248/404 in tissue culture. Biologically cloned derivatives of RSV cpts-248/404 virus that differed at position 4 possessed the same level of temperature sensitivity and exhibited the same level of replication in the upper and lower respiratory tract of mice, suggesting that heterogeneity at this position is not clinically relevant. The determination of the nucleotide sequence of the cpts-248/404 virus will allow evaluation of the stability of the eight mutations that are associated with the attenuation phenotype during vaccine production and following replication in humans.