Previous studies have shown that restraint stress modifies the development of allergic contact dermatitis (ACD). We hypothesized that the timing of restraint with respect to the chemical application influences restraint-induced modulation of ACD. Male BALB/c and C57BL/6 mice were sensitized on the flank on days I and 2 with 0.5% di-nitrofluorobenzene (DNFB) and challenged on the ear on day 6 with 0.25% DNFB. Mice were restrained for 2 hours prior to chemical application on day I only, on days 1 and 2, or on day 6 only. We assessed serum corticosterone to verify activation of the hypothalamic-pituitary-adrenal axis and ear swelling as a measure of the inflammatory/immune response. Restraint increased serum corticosterone 10 16 fold in both strains of mice. DNFB stimulated significant ear swelling in non-restrained mice. The ear swelling response was suppressed in DNFB-treated mice restrained for 2 hours on day 1 only, but was unchanged in DNFB-treated mice restrained on days 1 and 2. In contrast, mice restrained only on day 6 displayed significantly enhanced ear swelling, approximately 20% higher than levels measured in DNFB-treated, non-restrained mice. Administration of the glucocorticoid receptor antagonist, RU486, had little effect on the suppressed ear swelling response but did block the day 6 restraint-induced increase. These data demonstrate that timing of restraint affects the type and degree of modulation of chemical-induced ear swelling and that corticosterone may be more important in the enhanced ear swelling response than in the suppression of ear swelling.