The polymyxin antibiotics are inactivated in vitro by tissues because they are bound to phospholipids of cell membranes. A method that liberates bound drug in an active form from tissues permitted study of distribution and persistence in vivo. Studies using single injections in the rabbit showed that bound drug persists in liver, kidney, brain, heart, muscle, and lung for as long as 72 hr. Accumulation in tissue but not in serum was noted on repeated injection, with persistence for at least five days after seven daily injections. Free polymyxin B was detectable in liver, kidney, muscle, and brain and persisted for many days in muscle and brain. Free colistimethate was detectable in all tissues other than brain and persisted for many days in liver, kidney, and muscle. Colistimethate appears to be incompletely converted in vivo to the parent compound, colistin. These observations may account for differences in toxicity and chemotherapeutic efficacy of polymyxin Band colistimethate.