One hundred thirty-three ts mutants of influenza A/Udorn/72 virus were arranged into eight complementation groups, A-H, on Madin-Darby canine kidney (MDCK) monolayer cultures at the restrictive temperature of 40 degrees. The eight complementation groups, A-H, on MDCK cells corresponded to the eight recombination groups, A-H, on rhesus monkey kidney (RMK) cells, respectively, and this suggested that each MDCK complementation group represented one of the eight influenza A RNA gene segments. These ts viruses were used to identify the locus of the ts mutation in temperature-dependent host range (td-hr) mutants of the A/Udorn/72 virus. Sixteen of the 133 ts mutants exhibited distinct host (MDCK)-dependent restriction of plaque formation at 40 degrees but not at 34 degrees and were referred to as td-hr mutants. These 16 td-hr mutants were ts+ (not ts) on RMK cells but ts on MDCK cells. The td-hr mutants did not share a common lesion and the ts lesions were distributed among the eight complementation groups, A-H, when tested on MDCK cells. An analysis of one of the td-hr mutants indicated that an extrageneic RMK-dependent suppressor mutation did not account for the td-hr phenotype. These data suggested that a host-dependent ts mutation was responsible for the td-hr restriction of this mutant. Representation of td-hr mutations in each of the eight complementation groups indicates that the influenza A virus genome can undergo mutation leading to an altered host range in any of its eight RNA segments.