Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k(off)) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k(off) value increases from (1.4+/-0.2)x10(-4)s(-1), in the absence of the drug, to (9.5+/-1.2)x10(-3)s(-1), in the presence of 1.0x10(-2)M ibuprofen, at pH 7.0 and 10.0 degrees C. From the dependence of k(off) on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K(1)=(3.1+/-0.4)x10(-7)M, K(2)=(1.7+/-0.2)x10(-4)M, and K(3)=(2.2+/-0.2)x10(-3)M) were determined. The K(3) value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H=(2.6+/-0.3)x10(-3)M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow's site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.