We investigated the effects of interferon treatment on virus yield, protein synthesis, and the 2-5A system in vaccinia virus-infected HeLa, L929, and CV1 cells. Under the culture conditions used, vaccinia virus replication was relatively insensitive to the antiviral effects of interferon. In L929 and HeLa cells, interferon at 400 reference units (r.u.) per ml had little effect on viral protein synthesis, the virus-induced inhibition of host protein synthesis, or virus yield: 2,000 to 20,000 r.u./ml were required to inhibit these. Despite this, high levels (up to 5 microM) of 2-5A [ppp(A2'p)nA; n greater than or equal to 2] were found during vaccinia infection of all of these types of cells treated with 400 r.u. of interferon per ml, i.e., at interferon concentrations too low to inhibit significantly virus growth. High levels (up to 5 microM) were also found in non-interferon-treated HeLa cells (which have a high constitutive level of 2-5A synthetase) in which vaccinia virus replicates perfectly well. It can be concluded that high levels of 2-5A per se have no necessary antiviral effect on vaccinia virus in these systems. These results are in marked contrast to those obtained here and previously with encephalomyocarditis virus. For example, in HeLa cells less than 20 nM 2-5A accumulated, but virus replication was inhibited by 50 r.u. of interferon per ml (Silverman et al., Eur. J. Biochem. 124:131-138, 1982). The characteristic cleavage of rRNA by the 2-5A-dependent RNase was delayed relative to 2-5A accumulation in the vaccinia virus-infected cells. This delay was not the result of either defective 2-5A or of a stable virus-induced inhibition of the 2-5A-dependent RNase; 2-5A extracted from the cells had full biological activity when assayed by activation of the 2-5A-dependent RNase in cell extract, and the 2-5A-dependent RNase extracted from the vaccinia virus-infected cells was fully active in vitro. The basis for the delay remains to be determined. High levels of 2-5A were not observed when late (DNA synthesis-dependent) vaccinia transcription was inhibited by either cycloheximide or cytosine arabinoside. The only known activator of the 2-5A synthetase is double-stranded RNA. The presence of 2-5A therefore implies the natural occurrence of double-stranded structures in late viral RNA in intact vaccinia virus-infected cells.