The binding of nalidixic acid (NA) with human and bovine serum albumin (HSA and BSA) in buffer solution at pH 7.4 was investigated using circular dichroism (CD), UV absorption and fluorescence spectroscopy. Global analysis of multiwavelength spectroscopic data afforded the equilibrium constants of the most stable noncovalent drug/protein adducts of 1: 1 and 2:1 stoichiometry and their individual CD, UV absorption, and fluorescence spectra. The primary binding site of the drug was located in subdomain IIIA (Sudlow Site 11), whereas the secondary one was assigned to subdomain IIA. Conformational and CD calculations afforded the binding geometries. In the complexes, the fluorescence of the protein was strongly quenched by energy transfer and that of the drug was suppressed by electron transfer. Laser flash photolysis at 355 nm evidenced the formation of a radical pair consisting of a tyroxyl radical (lambda(max) = 410 nm) and a reduced nalidixate anion radical NA(2-center dot) (lambda(max) = 640 nm) with quantum yield of 0.4-0.5. Strong evidence was obtained that the process that involves Tyr411 in HSA (Tyr409 in BSA). A further transient with lambda(max) approximate to 780 nm observed in HSA was attributed to oxidation of the -(S200-S246)- bridge upon electron transfer to NA-*. Decay of the confined radical pairs occurred with rates approximate to 10(7) s(-1). Formation of covalent drug-protein adducts in mixtures irradiated at lambda(irr) > 324 nm was proved using HPLC with fluorescence detection.