In Brazil, where Leishmania chagasi causes endemic American visceral leishmaniasis (AVL), the spread and maintenance of human disease are attributed to canine reservoirs. However, despite measures directed toward the elimination of infected canines, the incidence of human disease continues to increase. To evaluate the role of infected canines in the acquisition of AVL by humans, we undertook a controlled intervention study in three similar, but isolated, valleys of Pancas, Espírito Santo, Brazil. In the two experimental (intervention) valleys, infected dogs were eliminated whereas in the control valley, seropositive canines remained untouched. During the 12-month study period, human seropositivity rates, as measured by dot enzyme-linked immunosorbent assay, increased from 15% to 54% in the intervention valleys and from 14% to 54% in the control valley. The elimination of infected canines in the intervention valleys did not result in a statistically significant difference between the incidences of human serological conversion in the intervention and control valleys at either 6 (20% and 22%, respectively; P = .5961) or 12 months (26% and 27%, respectively; P = .9442). The role of humans as a significant reservoir for AVL is proposed as an explanation for the study results.