Abstract: Pumiliotoxins (PTX) A, B, and 251D, members of a new class of indolizidine alkaloids isolated from the skin of poison frogs of the family Dendrobatidae, inhibit Ca2+‐ATPase activity in sarcoplasmic reticulum vesicles from frog and rat hind‐limb muscles. PTX‐B and PTX‐A appear to be relatively specific inhibitors of Ca2+‐ATPase; PTX‐A is much less potent than PTX‐B. PTX‐251D is a potent inhibitor of Ca2+‐ATPase, and was also found to inhibit Na+, K+, and Mg2+‐ATPases in rat brain synaptosomes. Caffeine and verapamil, two drugs known to affect calcium translocation, are very weak inhibitors of the Ca2+‐ATPase. The K, values for inhibition of the Ca2+‐ATPase of rat and frog sarcoplasmic reticulum by PTX‐B were comparable and ranged between 22 and 36 μM. Inhibition of calcium‐dependent ATPase in sarcoplasmic reticulum by pumiliotoxin‐B is noncompetitive with calcium and is not readily reversible. Based on structure‐activity profiles, it is concluded that inhibition of Ca2+‐ATPase by the indolizidine alkaloids is responsible for the alkaloidelicited prolongation of twitch in intact muscle. Copyright © 1982, Wiley Blackwell. All rights reserved