A remarkable diversity of bioactive lipophilic alkaloids is present in the skin of poison frogs and toads worldwide. Originally discovered in neotropical dendrobatid frogs, these alkaloids are now known from mantellid frogs of Madagascar, certain myobatrachid frogs of Australia, and certain bufonid toads of South America. Presumably serving as a passive chemical defense, these alkaloids appear to be sequestered from a variety of alkaloid-containing arthropods. The pumiliotoxins represent a major, widespread, group of alkaloids that are found in virtually all anurans that are chemically defended by the presence of lipophilic alkaloids. Identifying an arthropod source for these alkaloids has been a considerable challenge for chemical ecologists. However, an extensive collection of neotropical forest arthropods has now revealed a putative arthropod source of the pumiliotoxins. Here we report on the presence of pumiliotoxins in formicine ants of the genera Brachymyrmex and Paratrechina, as well as the presence of these ants in the stomach contents of the microsympatric pumiliotoxin-containing dendrobatid frog, Dendrobates pumilio. These pumiliotoxins are major alkaloids in D. pumilio, and Brachymyrmex and Paratrechina ants now represent the only known dietary sources of these toxic alkaloids. These findings further support the significance of ant-specialization and alkaloid sequestration in the evolution of bright warning coloration in poison frogs and toads.