An immediate reaction was investigated during repeated challenge testing for contact hypersensitivity to dinitrofluorobenzene (DNFB) in BALB/c mice. The mice were sensitized to DNFB on back skin and repeatedly challenged with the same hapten on the left ear at 1 week intervals. The ear after the 5th challenge showed biphasic responses which consisted of an immediate and a delayed-type reaction. The reactions were hapten specific. Mast cell-deficient WBB6F1 W/WV mice did not show any immediate reaction, while congenic normal mice showed both immediate and delayed-type reactions. Histologically, numerous dermal mast cells were found in the left ear of repeatedly challenged BALB/c and WBB6F1 normal mice, while there were few mast cells in the ear of WBB6F1 W/WV mice. Anti-DNP IgE antibodies were detected in BALB/c, WBB6F1 normal and W/WV mice after repeated challenge with DNFB. Intradermal injection of anti-IgE antibodies in the repeatedly DNFB-challenged ear elicited an immediate reaction. These results suggest that immediate contact hypersensitivity develops through the production of anti-DNP IgE antibodies and an increase in dermal mast cells after repeated challenge with DNFB.