Influenza A/Hong Kong/68-ts-1[E] virus is a temperature-sensitive mutant developed for use as a live virus vaccine (B. R. Murphy, E. G. Chalhub, S. R. Nusinoff, J. Kasel, and R. M. Chanock, J. Infect. Dis. 128:479--487, 1973). This virus and temperature-sensitive recombinants derived by mating it with A/Udorn/72, A/Georgia/74, or A/Victoria/75 wild-type virus have been administered to volunteers in clinical trials on the assumption that the ts-1[E] temperature-sensitive genetic lesions on a polymerase gene (P3) and on the nucleoprotein gene (NP) would determine a satisfactory and reproducible level of attentuation regardless of the genetic constitution of ts-1[E] recombinants at other loci (B. R. Murphy, D. D. Richman, S. B. Spring, and R. M. Chanock, Postgrad. Med. 52:381--388, 1976). In this paper, the parental origin of genes in the ts-1[E] recombinants was determined by using the technique of polyacrylamide gel electrophoresis of virion ribonucleic acid segments in the presence of a denaturing agent (urea). When tested in individuals who lacked immunity to hemagglutinin antigen, attenuation of the ts-1[E] recombinants appeared to correlate with inheritance of the ts-1[E] temperature-sensitive genes at the P3 and NP loci and with the level of preinfection neuraminidase immunity. There was no evidence that other genes from the ts-1[E] donor virus played a role in attenuation.