Rodents collected from the Venezuelan llanos (plains) during field studies of viral hemorrhagic fever were tested for evidence of hantavirus infection. Hantavirus antibody was found in one (7.7%) of 13 Oryzomys bicolor, one (3.4%) of 29 Rattus rattus, 10 (6.0%) of 166 Sigmodon alstoni and one (2.2%) of 45 Zygodontomys brevicauda. Hantavirus-specific RNA was detected in lung tissues from four antibody-positive rodents: two S. alstoni from Portuguesa State and one S. alstoni each from Cojedes and Barinas States. A hantavirus isolate (herein identified as VHV-574) was recovered from lung tissue from a hantavirus RNA-positive S. alstoni collected from Portuguesa State. The results of serological tests and analyses of small and medium RNA segment nucleotide sequence data indicated that VHV-574 represents a novel hantavirus (proposed name 'Caño Delgadito') that is distinct from all previously characterized hantaviruses. The results of analyses of nucleotide sequence data from the four hantavirus RNA-positive S. alstoni suggested that Caño Delgadito virus is widely distributed in the Venezuelan llanos.