The reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) with thiols is sensitive to daylight, in particular to ultraviolet radiation at wavelengths around 325 nm. Exposure to light at the absorbance maximum of the yellow product (the thionitrobenzoate ion) at 410 nm had no effect on the reaction. The light-sensitive species is apparently the DTNB, because a spectral-irradiation experiment showed that the wavelength of light that produced the maximum rate of absorbance change coincided with the peak absorbance of DTNB, and it was well separated from the thionitrobenzoate absorbance peak. Ascorbate is ineffective as a stabilizer and can produce an apparent increase in the rate of DTNB destruction. In a practical example we found the light interference to be severe when hydrolysis of propionylthiocholine by plasma cholinesterase (EC 3.1.1.8) was measured after a 20-min incubation. The apparent cholinesterase activity in clear glass or plastic tubes exposed to diffuse daylight could be decreased to 25% of the value obtained for samples in light-excluded tubes. We recommend the reaction be carried out in artificial room light, with total elimination of daylight, because window glass does not sufficiently attenuate 325-nm wavelength irradiation.