The antitumor agent caracemide [N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea] has been observed to induce signs of cholinergic activation in both animal and human studies at high clinical and toxic levels. The present study was designed to further characterize the ability of caracemide to inhibit cholinesterase both in vivo and in vitro. Caracemide produced a time-dependent inhibition of purified acetylcholinesterase, pseudocholinesterase and rat brain homogenate cholinesterase with IC50 values of 0.60, 0.55 and 17.8 microM, respectively, at the maximal time point of inhibition. Analysis of Lineweaver-Burke plots derived from inhibition of brain homogenates revealed predominantly competitive type inhibition at both initial and maximal incubation times. Intravenous administration of caracemide to rats resulted in a dose-dependent inhibition of brain cholinesterase activity, with the greatest inhibition occurring in the cortex.