The tropical forest canopy and litter differ in physical structure, resource availability, and abiotic conditions. We used standardized bait experiments in the canopy and litter of four neotropical tree species to explore how these differences shape the behavior, morphology, and diversity of ant assemblages. Ant activity (biomass at a bait after 32 min) was higher in the canopy, and higher on protein baits than carbohydrate baits. Aggressive bait defense occurred more frequently in the canopy (60%) than in the litter (32%), but was not associated with tree species or bait type in either habitat. The median size of workers of species in the canopy and litter was nearly identical, but body size distribution was unimodal in the canopy and bimodal in the litter. The colony size of the most aggressive species was an order of magnitude larger in the canopy. Species richness at a bait was relatively uniform across tree species and habitats. Litter and canopy shared no species, but overlap among tree species was three times higher in the litter assemblages. Litter assemblages showed less activity, less interference, less differentiation across the landscape, and different size distributions than canopy assemblages. The canopy and litter templets subsume a number of environmental gradients that combine to shape ant community structure.