The absorption spectrum of indocyanine green depends on the nature of the solvent medium and on the dye concentration. Binding to plasma proteins causes the principal peaks in the absorption spectrum to shift about 25 nm toward the higher wavelengths. The much greater influence on the spectrum of the dye concentration results from progressive aggregate formation with increasing concentration. Indocyanine green solutions therefore do not follow Lambert-Beer's law above 15 mg-I-1 (in plasma). Indocyanine green solutions in plasma and concentrated (1,000 mg-I-1) solutions in distilled water are stable for at least 4 h. In long-term experiments the optical density of indocyanine green solutions in plasma as well as in distilled water generally diminishes, even in the dark. On the 7th day a new absorption maximum starts to appear at gamma=900 nm, possibly caused by further aggregate formation leading to much larger particles. Spectral stabilization after injection of a concentrated solution into the blood is most rapid when the dye is dissolved in distilled water. Spectral stabilization slows down with decreasing temperature. As rapid spectral stabilization is essential in quantitative dye dilution studies, the practice of adding a albumin and/or isotonic saline solution to the injectate should be discontinued. When a 10 g-1(-1) aqueous solution of indocyanine green is used, spectral stabilization takes less than 1.5 a (at 37 degrees C), which is sufficiently fast for almost any application.