In some populations of the harvester ant Pogonomyrmex californicus, founding queens can form pleometrotic associations and subsequently develop a division of labor that lasts until the emergence of the first workers. In associations where this occurs, one or more queens specialize on reproduction and the others focus on tasks usually performed by the worker caste, such as nest maintenance and foraging. Evidence from other hymenopteran systems suggests that these types of behavioral differences are correlated with divergent hormone titers, which can be influential on behavior directly or indirectly via activational or organizational effects. To determine if the behavioral diphenism seen in pleometrotic foundations is associated with hormonal differences, we measured juvenile hormone and ecdysteroid content in queens that were either primarily egg layers or foragers. We found significant differences in the JH titers of the two groups, suggesting that JH influences behavioral phenotype. This link is supported by hormonal data from haplometrotic populations of the same species, where single queens are characterized by a behavioral sequence in which reproduction is followed by foraging. Corresponding associations between hormone levels, sequential founding behavior and social division of labor give credence to the idea that modification of reproductive endocrine regulators provided a basis for the evolution of caste specialization in social insects.