The diaminopyrimidine derivatives trimethoprim (TMP), pyrimethamine (PMA) and 2,4-diaminopyrimidine (2,4-DAP) are found to react readily and efficiently in an aqueous solution with p-benzoquinone (p-BQ) to form a colored product with an absorption optimum wavelength of about 500 nm. The optimum reaction time, pH, temperature, solvent, and [p-BQ] are determined by separate trials. These conditions are confirmed by a MultiSimplex optimization method. The molar absorptivities of the TMP, PMA, and 2,4-DAP reaction products at 500 nm are 10,830, 10,650, and 9660 l mol(-1) cm(-1), respectively. TMP shows a linear range between 5 and 100 mg/l while PMA and 2,4-DAP exhibit linearity between 15 and 75 mg l(-1) and 5 and 30 mg l(-1), respectively. There is some specificity to this reaction; 2-aminopyrimidine does not react. Under the optimum conditions, sulfamethoxazole (SM) reacts rather poorly with a molar absorptivity of about 110 l mol(-) cm(-1). Using p-BQ, TMP in a pharmaceutical sample can be determined in the presence of SM using derivative spectrophotometry. The TMP-p-BQ reaction is adaptable for flow injection analysis.