Using a contact hypersensitivity model in BALB/c mice we have previously been able to show that Corynebacterium parvum or C. parvum serum (C.p.s.) of mice treated 24 hr before with C. parvum inhibited the suppressor T-lymphocyte (Ts-cell) response induced by epicutaneous antigen overload with 2,4-dinitrofluorobenzene (DNFB) or by i.v. injection of 2,4-dinitrobenzene sulfonic acid (DNBSO3) without inhibition of the T effector cell response (TDH-cell). In the present investigation we further analyzed the factor responsible for Ts-cell inhibition. Treatment of the C.p.s. with sheep-antimouse interferon (IFN) globulin neutralized its Ts-cell inhibitory effect. Intravenous (i.v.) injection of a crude mouse fibroblast IFN (340 U per mouse) 2 hr after i.v. application of a dose of DNBSO3 inducing tolerance had a similar Ts-cell inhibitory effect as observed with C.p.s. Injection of an electrophoretically pure alpha- and beta-IFN preparation (1000 U per mouse) increased contact sensitivity in mice sensitized with an antigen overload and inhibited the induction of Ts-cells by DNBSO3 i.v. This result is highly suggestive that the Ts-cell inhibitory factor in serum of C. parvum-treated mice is IFN and it shows that Ts-cells as compared to TDH-cells are susceptible to the inhibitory effect of highly purified IFN. This finding suggests that IFN may be an important immunoregulatory factor of delayed hypersensitivity not only in contact allergy but also in bacterial and viral defense.