The incorporation of imipramine into isolated rat peritoneal mast cells was fluorometrically studied in vitro. The binding of imipramine was characterized by the following features: 1. it took place in two phases, the first of which was very rapid, 2. it increased with increasing exogenous concentration in a sigmoidal fashion, 3. it increased with increasing exogenous pH, 4. it was dependent on neither temperature nor calcium ions in the medium nor did the binding occur specifically to mast cells but also to leucocytes, 5. it was reduced by several imipramine derivatives, tricyclic antidepressants and 5‐hydroxytryptamine at high concentrations, 6. about 40 % of the cell‐bound drug was easily washed out; the remaining 60 % represented a level approached after prolonged washing and seemed to be fairly tightly bound, 7. the subcellular location of imipramine in mast cells was different from that of 5‐hydroxytryptamine, indicating a nongranular binding of the drug. It is concluded that imipramine binds to mast cells in a passive manner and is associated with the cell membrane where it seems to be bound to at least 2 different sites. At low concentrations imipramine is probably bound to sites specifically influencing the operativeness of the amine carrier. Higher concentrations of imipramine results in binding to nonspecific sites resulting in impairment of the structure and function of the cell membrane.