In the pH range 6-9, human serum albumin undergoes a conformational change termed the neutral-base (N-B) transition. Recently, it has been shown that the N-B transition causes enhanced binding at the warfarin-binding site (site I). The present study used fluorescence and equilibrium dialysis to investigate the effects of the N-B transition, chloride, calcium and fatty acids on the specific binding sites I and II on human serum albumin. The effect of the N-B transition of human serum albumin provides a further distinction between site I and II binding characteristics. The N-B transition of albumin caused a change in conformation at site I which resulted in increased binding of drugs and fluorescent probes at this site, whereas there was no effect on acidic drug binding at site II. These effects on site I and II are qualitatively similar to those induced by fatty acids (increased drug binding at site I and no change at site II). However, the effects of increasing pH and fatty acids were additive, showing that they were caused by two different conformational changes. The effect of Cl- on site I binding was pH-dependent and was abolished by the presence of fatty acid. Ca2+ reduced the fluorescence of site I probes but had no effect on a site II fluorescent probe. Effects of pH were also investigated with drugs not binding to site I or II. Increasing pH caused a decrease in binding to indomethacin, increases in binding of L-tryptophan, tolmetin and quinidine and no change in the binding of salicylic acid, diflunisal and phenytoin.