Ascertaining the aqueous solubility of compounds is important in the selection of drug candidates. We describe a radiotracer method for estimating water solubility of compounds that are soluble in dimethyl sulfoxide (DMSO). Various volumes of DMSO, saturated with 127 I-labeled compound and spiked with the corresponding 125 I-labeled derivative, are mixed in de-ionized water and the tubes centrifuged to remove insoluble material. Since (i) the iodine-127 and iodine-125 compounds have the same solubilities and are equally distributed in the DMSO-water solution, and (ii) the nonradioactive compound is accurately weighed, dissolved in a known volume of DMSO, and then further diluted as required, the concentration of compound in solution can be calculated and plotted as a function of the DMSO-to-water ratio. Water solubility of the compound is then determined by extrapolation of the linear fit of data points to zero DMSO. As proof of the methodology, 5-iodo-2 0 -deoxyuridine (IUdR) and 2-iodo-8-methyl-8H-quino[4,3,2-kl]acridine (IMAc), water-soluble compounds, were assessed using 125 IUdR and 125 IMAc, respectively. The solubility values obtained by the radiotracer method were similar to those acquired by spectrophotometry. Values calculated for several water-insoluble compounds indicate that the radiotracer method can accurately quantify the solubility of low-molecular-weight compounds (1000-2000 Da) in the pg-ng/ml range.