Effects of pertussis toxin (PT) on the unresponsiveness of delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were examined in mice. DTH cannot be detected in mice injected iv with 1 X 10(9) SRBC. This unresponsive state was broken by 1 microgram of PT given iv to mice 1 or more days, even 21 days, after SRBC-injection and replaced by the development of high and persistent DTH responses with several days' lag after PT treatment. The restored DTH response was accompanied by the appearance of circulating DTH-mediating cells. In the non-PT-treated mice, DTH-mediating cells, which conferred DTH in syngeneic naive recipient mice by systemic transfer, were found in the spleen but not in the blood, whereas in the PT-treated mice, they were found both in the spleen and in the blood. On the other hand, the induction or the function of splenic suppressor T cells (Ts) for DTH in mice injected iv with 10(9) SRBC was inhibited by PT treatment. DTH-Ts, which appeared in the spleen around 4 days after SRBC injection and inhibited the induction of DTH response to SRBC in the syngeneic cyclophosphamide-pretreated recipient mice, failed to be induced in mice given PT 1 day after SRBC injection, and their ability was reduced when they were treated in vitro with PT immediately before transfer. These results demonstrate the possibilities that the reinforced migration of DTH-mediating cells from spleen to blood by PT is involved in the mechanisms by which PT break the unresponsiveness of DTH and that the interference with the induction or the function of DTH-Ts by PT may also be partly involved in the mechanisms.