A total of 1,500 small mammals were collected and tested for antibodies cross-reactive to Sin Nombre virus (Hantavirus: Bunyaviridae) at 89 sites in a 1,600 km2 study area of southern Florida. More than 95% of the 123 seropositive animals were cotton rats (Sigmodon hispidus), suggesting infection by Black Creek Canal Virus, although seroreactive Rattus rattus (5 of 294) and Peromyscus gossypinus (1 of 39) also were captured. Crude seroprevalence in S. hispidus was 11%. Seroprevalence increased with body size and was more common in male (18%; n=451) than in female (6%; n=593) cotton rats. Infection within S. hispidus populations was widespread throughout the study area. Prevalence ranged from 0% to 60% at sites where more than five cotton rats were sampled but was not only a function of sample size. Sites with seropositive cotton rats were geographically clustered compared with sites with no seropositive cotton rats. Clustering was not due to the spatial distribution of sites with few animals, season of collection, or sex bias of animals captured at these sites. However, sites with no seropositive animals had an excess of animals in the intermediate size class (60-99 g) and a deficit of the largest and smallest animals. These data suggest that population structure within the habitat mosaic may play a significant role in the spatial distribution of hantavirus infection in local populations of reservoir species.