Binding of chlorpromazine (CPZ) with human hemoglobin has been studied by equilibrium dialysis and fluorescence quenching. Results of equilibrium dialysis experiment when analysed by Hill plot revealed that the binding was positively cooperative with overall affinity constant K = 3.8 × 103 M-1. CPZ quenched the fluorescence of hemoglobin and the analysis of the quenching data by Stern-Volmer equation indicated two types of quenching process, namely, dynamic and static quenching. Dynamic quenching constant was measured from the decay of fluorescent life time of tryptophans of hemoglobin in presence of CPZ. Static quenching constant concerned with the ground state complex formation between CPZ and hemoglobin was found to be 5 × 103 M-1. Almost all the tryptophans of hemoglobin were found to be accessible for CPZ to interact with. Oxygen was found to be released when CPZ was added to hemoglobin. Extent of release of oxygen depends on the D P ratio of CPZ(D): hemoglobin(P). © 1990.