For the first time, an aqueous solution of 32-nm gold nanoparticles (GNPs), to which Nile red (NR) has been noncovalently adsorbed, has been used for sensing thiols. The as-prepared NRGNPs fluoresce weakly as a result of fluorescence resonance energy transfer between NR and the GNPs. The fluorescence of a solution containing NRGNPs at pH 4.0 increases upon the addition of thiols, but not when amines, acids, alcohols, bovine serum albumin, or hemoglobin are added. This phenomenon allows for the selective determination of thiols such as cysteamine and homocysteine, which have limits of detection of 10.2 and 10.9 nM, respectively, at a signal-to-noise ratio of 3. Interestingly, we have found that the excitation (lambda(ex) = 480 nm), emission (lambda(em) = 610 nm), and mass spectra (m/z 282) of the substance that desorbs from the GNPs in the presence of thiols are different from those of NR (lambda(ex) = 580 nm; lambda(em) = 652 nm; m/z 318), which indicates that a new product forms. When simultaneously conducting fluorescence and colorimetric assays, the selectivity of this approach further improves because at pH 4.0, the color of the NRGNPs does not change in the presence of negatively charged thiols, (e.g., N-(2-mercaptopropionyl)glycine), but changes from maroon to purple and lavender in the presence of neutral thiols (e.g., 3-mercapto-1,2-propanediol) and positively charged thiols (e.g., cysteamine), respectively, as a result of aggregation. This feature allows the types of thiols to be determined at concentrations >1.0 and 0.1 microM by the naked eye and by UV-vis absorption, respectively. Depending on the rate at which the NRGNP color changes, reduced glutathione (slow) is readily distinguishable from oxidized glutathione (no aggregation and no displacement) and from cysteine and homocysteine (fast).