In 1995, an outbreak of hantavirus pulmonary syndrome occurred in the central Paraguayan chaco. The primary reservoir of the virus, Laguna Negra virus, was identified as the vesper mouse, Calomys laucha. Over a 15-month period, we collected 1,090 small mammals at 12 locations representing 4 habitats common in the central Paraguayan chaco. Calomys laucha was common in agricultural habitats and uncommon in the native forest habitat. Populations of C. laucha were greater during the dry season months and declined during the wet season. A total of 643 small mammals were tested for antibodies cross-reactive to Sin Nombre virus. All of the antibody-positive animals were C. laucha (crude antibody prevalence ratio 12.1% [25 of 206]). Antibody prevalence ratio increased with body size and was more common among male (18%; n = 115) than among female (4%; n = 96) vesper mice. Antibody prevalence ratio was highest among animals from cropland habitats (18%; n = 72), followed by thorn scrub (13%; n = 46) and pastureland (7%; n = 81) and may be positively correlated to the proportion of C. laucha in the small mammal community. These data suggest that community-level dynamics, in addition to population-level dynamics, may be involved in the transmission of the virus through natural populations of vesper mice.