Forty-eight Sigmodon alstoni (Alston's cotton rat) were inoculated with Caño Delgadito (CDG) virus to extend our knowledge and understanding of the natural host relationships of the hantaviruses indigenous to the Americas. Infectious CDG virus was recovered from oropharyngeal secretions, urine, or solid tissues of nine of 12 animals killed on day 9 post-inoculation (PI), 14 of 24 animals killed on day 18 or 27 PI, and none of 12 animals killed on day 54 PI. In addition, virus-specific RNA was detected in the kidneys of six of the 12 animals killed on day 54 PI, and adult cotton rats inoculated with the kidneys of four animals killed on day 54 PI developed antibody to CDG virus. Collectively, the results indicate that CDG virus can establish lengthy (perhaps lifelong) infections in Alston's cotton rat and thus support the concept that S. alstoni is the principal host of CDG virus.