A site-oriented study of nine non-steroidal anti-inflammatory drugs (NSAIDs), suprofen, ibuprofen, diclofenac sodium, pirprofen, flurbiprofen, ketoprofen, phenylbutazone, oxyphenbutazone, and ketophenylbutazone, on human serum albumin (HSA) was carried out at pH 7.4 by various direct and indirect methods to gain insight into the high affinity binding sites of NSAIDs. The binding was determined by equilibrium dialysis, circular dichroism and fluorescence methods in order to strengthen the results. Irrespective of the method used, close agreement between the binding parameters obtained by the different methods was obtained. A site-oriented description, as revealed by the fluorescent probe displacement method, suggests that site II and site I probes bound to HSA were selectively displaced by NSAIDs with a carboxyl group and without a carboxyl group, respectively. The absence of a carboxyl group of the NSAIDs changed the binding site from site II to site I. Different binding models describing the competitive and independent binding of a NSAID and a site-specific probe bound simultaneously to HSA further describe the respective high affinity binding sites for NSAIDs, thus underscoring the necessity of a carboxyl group in order for a NSAID to bind to site II. The presence of tyrosine, lysine and histidine amino acid residues in the binding of carboxyl group-containing NSAIDs to HSA was evident, whereas tryptophan is believed to take part in the binding of non-carboxyl group-containing NSAIDs. The present findings support the proposal that two separate primary binding sites exist for different NSAIDs; hence, an attempt to correlate the present results with those in literature has been made.