The frequency of astrovirus infection in 456 Chilean children with diarrhea was determined by enzyme-linked immunosorbent assay, reverse transcriptase PCR, and cell culture. Astrovirus was detected in 16.5% of rotavirus-negative and 7% of rotavirus-positive samples obtained from emergency rooms or hospitals and in 11% of samples from day care centers. HAst-1 was the predominant serotype identified.