In order to measure turnover rates of the noradrenergic, dopaminergic, and serotonergic transmitter systems in rat brain, a method was developed by which norepinephrine, dopamine, and 5-hydroxytryptamine, and their main metabolites 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, could be measured simultaneously. High-performance liquid chromatography in the reversed-phase mode, including ion pairing, separated the transmitters and their metabolites well. By means of enzymatic hydrolysis of the sample prior to chromatography, it was also possible to measure the conjugated forms of the metabolites. Since there was no prepurification step, the hydrolysed supernatants of tissue homogenates were injected directly into the chromatographic system; additional selectivity tests were necessary. Peak identification was confirmed by comparison of hydrodynamic voltagrams and capacity factors at different pH values of the mobile phase of the components in the sample and the standard solution. The method is demonstrated by analysing mediobasal hypothalamic tissues of probenecid-treated rats.