The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.