Mammalian defensins are cationic, antimicrobial peptides that play a central role in innate immunity. The peptides are composed of three structural subfamilies: α-, β-, and θ-defensins. θ-defensins are macrocyclic octadecapeptides expressed only in Old World monkeys and orangutans and are produced by the pair-wise, head-to-tail splicing of nonapeptides derived from their respective precursors. The existence of three active θ-defensin genes predicts that six different RTDs (1-6) are produced in this species. In this study, we isolated and quantified RTDs 1-6 from the neutrophils of 10 rhesus monkeys. RTD-1 was the most abundant θ-defensin, constituting ~50% of the RTD content; total RTD content varied by as much as threefold between animals. All peptides tested were microbicidal at ∼1 μM concentrations. The contribution of θ-defensins to macaque neutrophil antimicrobial activity was assessed by analyzing the microbicidal properties of neutrophil granule extracts after neutralizing θ-defensin content with a specific antibody. θ-defensin neutralization markedly reduced microbicidal activities of the corresponding extracts. Macaque neutrophil granule extracts had significantly greater microbicidal activity than those of human neutrophils, which lack θ-defensins. Supplementation of human granule extracts with RTD-1 markedly increased the microbicidal activity of these preparations, further demonstrating a prominent microbicidal role for θ-defensins.