We have previously reported that T1026, a temperature-sensitive (ts) noncytocidal mutant of VSV, and its ts revertant, T1026-R1, are nonconditional mutants in the VSV function "P" for the inhibition of total protein synthesis (viral plus cellular) in infected cells (C. P. Stanners, A. M. Francoeur, and T. Lam, 1977, Cell 11, 273-281; C. P. Stanners, S. Kennedy, and L. Poliquin, 1987, Virology 160, 255-258). We have also shown that P- mutants such as these are superior interferon inducers relative to their parental P+ wild-type virus, HR, and that P- mutants may be distinguished from P+ virus using the plaque interferon production of PIF assay. (A. M. Francoeur, T. Lam, and C. P. Stanners, 1980, Virology 105, 526-536). In order to carry the analysis of VSV P function further, a number of independent mutants in the VSV P function are required. We show here that the PIF assay may be used to isolate spontaneously occurring interferon-inducing mutants (PIF+ mutants) from wild-type VSV (PIF- virus) populations. About one-half of the PIF+ mutants isolated with the PIF assay were found to have alterations in the VSV P function. As well as mutants that were defective for the inhibition of total protein synthesis, the assay yielded a new class of VSV P function mutants which appear to inhibit protein synthesis more severely than does P+ virus. The majority of newly isolated PIF+ mutants was also found to be temperature sensitive for growth. The ts phenotype, however, could be reverted for most PIF+ mutants with little effect on the PIF or P phenotype. These findings show that interferon induction and P function are related functions of VSV; this fact has allowed the isolation of a repertoire of mutants with widely varying P function.