Cells lacking thymidine kinase (tk) have been reported to fail to respond to the action of interferon (IFN). While some laboratories have confirmed this observation, others have failed to do so. We studied the effect of IFN on four freshly isolated tk- lines of mouse L cells infected with mengovirus. In all cases, normal antiviral activity was induced. The antiproliferative activity of IFN was studied using the parental L cell line, a tk- derivative, and a tk- (tk+) subline into which the tk gene of herpes simplex virus was introduced. All three lines had a doubling time of about 20 h. In all cases, 2,000 U/ml of IFN increased this time to about 50 h. In contrast to the above results, an IFN-sensitive mutant of mengovirus (is-1) grew much better in protected tk- cells than in protected normal cells. This phenomenon appears to be dependent on the fact that tk- cells were routinely maintained in medium containing 5-bromodeoxyuridine (BUDR). In the absence of this drug, the virus behaved normally. The implications of this observation are discussed.