We studied the effect of one application of compound 48/80 to the conjunctiva of the rat on the response to a subsequent challenge. Rats treated once showed conjunctival edema and marked degranulation of conjunctival mast cells. A second exposure to compound 48/80, applied after an interval of 24 h, produced slight clinical effects but had marked effects on conjunctival mast cells. Approximately 90% of the mast cells could no longer be observed by light microscopy. As the interval between initial challenge and rechallenge was increased, the clinical response returned, mast cells were again observable in normal numbers, and the extent of degranulation returned to approximately 50%. After an interval of 7 days, rats responded clinically as they had to the first application of compound 48/80. Although the number of mast cells in the conjunctiva was normal and extensive degranulation occurred, exocytosis was not observed. Thus some effects of the first application of compound 48/80 persisted for at least 1 week. Whether the effects observed depend upon the continued presence of compound 48/80 in the mast cell or granule membrane remains to be determined.