A comparison was made of the inhibitory effect of amethopterin and 3′,5′-dichloro-amethopterin (DCM) on the incorporation in vivo of formate-C14 into the acid-soluble adenine of mouse spleen infiltrated with leukemia L1210. Inhibition was studied as a function of dose and of time interval between the administration of the drug and that of formate. With a short interval of 20 minutes, small doses of amethopterin (0.32 mg/kg) and of DCM (0.64 mg/kg) produced considerable and about equal inhibition. The action of DCM was more transitory than that of amethopterin, so that large doses of DCM were required to cause prolonged inhibition. The dose of DCM that depressed formate incorporation during 24 hours was found to be of the same order as the dose which produces maximum median survival times in leukemic mice on daily treatment. The shorter duration of the effect of DCM as compared with that of amethopterin correlates with its lower toxicity in mice. Very large doses of citrovorum factor were able to block the inhibitory effect of the folic acid antagonists on formate incorporation in vivo. These findings are discussed with respect to mechanism of action and chemotherapeutic implications.