In order to establish whether a relationship exists between the activity of hydroxylating drug metabolizing enzymes and the activation of CCl₄, the study of the irreversible binding of the ¹⁴C from ¹⁴CCl₄ to microsomal lipids was undertaken under differing conditions of drug metabolism. Liver and kidney microsomes have greater ability to activate CCl₄ than their respective mitochondrial or 105,000 g supernatant fractions. In adrenals, an unexpectedly high CCl₄ activating activity was found not only in microsomes but also in mitochondria. The CCl₄ activating ability was significantly higher than in controls in the following experimental conditions: in SKF 525A treated rats; in adrenalectomized rats; and in 72 hr starved rats. Activation was not significantly changed in castrated, Sch 5706 treated or aminopyrine treated rats and was decreased in female, DPEA treated or nicotinamide treated rats. The results are discussed in relation to a possible activation of CCl₄ occurring during the reduction of the CCl₄-cytochrome P-450 complex mediated by cytochrome P-450 reductase or arising during the interaction of CCl₄ with reduced cytochrome P-450.