Previous studies of the division of labor in colonies of eusocial Hymenoptera (wasps and bees) have led to two hypotheses regarding the evolution of juvenile hormone (JH) involvement. The novel-or single-function hypothesis proposes that the role of JH has changed from an exclusively reproductive function in primitively eusocial species (those lacking morphologically distinct queen and worker castes), to an exclusively behavioral function in highly eusocial societies (those containing morphologically distinct castes). In contrast, the split-function hypothesis proposes that JH originally functioned in the regulation of both reproduction and behavior in ancestral solitary species. Then, when reproductive and brood-care tasks came to be divided between queens and workers, the effects of JH were divided as well, with JH involved in regulation of reproductive maturation of egg-laying queens, and behavioral maturation, manifested as age-correlated changes in worker tasks, of workers. We report experiments designed to test these hypotheses. After documenting age-correlated changes in worker behavior (age polyethism) in the neotropical primitively eusocial wasp Polistes canadensis, we demonstrate that experimental application of the JH analog methoprene accelerates the onset of guarding behavior, an age-correlated task, and increases the number of foraging females; and we demonstrate that JH titers correlate with both ovarian development of queens and task differentiation in workers, as predicted by the split-function hypothesis. These findings support a view of social insect evolution that sees the contrasting worker and queen phenotypes as derived via decoupling of reproductive and brood-care components of the ancestral solitary reproductive physiology.behavioral development ͉ division of labor ͉ methoprene ͉ worker polyethism