A detailed dose-response curve has been established for induction of contact hypersensitivity (CH) in mice with trinitrochlorobenzene (TNCB). It was determined that in BALB/c, CBA/J, and C57BL/6 mice, the dose required to sensitize via epicutaneous application was between 1 and 10 micrograms TNCB. When doses of hapten of 200 micrograms or greater were painted on abdominal skin, CH responses were induced which were only marginally greater than responses induced by sensitizing doses of hapten in the 10-50-micrograms range, implying that no further dose-response relationship exists beyond 50 micrograms of hapten. However, in companion experiments, in which panels of mice were pretreated with cyclophosphamide, it was determined that sensitizing doses of hapten in excess of 50 micrograms induced both CH and concomitant induction of down-regulation of CH. Thus, at 200 micrograms or higher doses of TNCB, CH responses of cyclophosphamide pretreated mice were invariably more intense than in their untreated, hapten-painted cohorts. In the animals pretreated with cyclophosphamide, it was possible to see that a dose-response relationship continued to exist between the amount of epicutaneously applied hapten over a 200 micrograms to 14 mg range and the intensity of the CH induced. We conclude that the optimal dose for immunizing mice epicutaneously with TNCB is between 10 and 50 micrograms. This is considered optimal since animals sensitized in this manner display no evidence of concomitant down-regulation of their CH responses.