Rotavirus, adenovirus, norovirus, sapovirus and astrovirus are considered to be significant global enteropathogens associated with sporadic cases and outbreaks of acute gastroenteritis. Therefore, a rapid and sensitive assay is preferred to screen for the presence of these viruses in diarrheal fecal specimens. In a previous study, we developed a reverse transcription single-round multiplex polymerase chain reaction (RT-smPCR) assay for the simultaneous detection of norovirus (genogroup I, genogroup II), sapovirus and astrovirus in fecal specimens (Yan et aL, 2003). Recently, we developed another RT multiplex PCR for one-step amplification of all subgenera A to F adenoviruses, and group A and C rotaviruses. In this study, a total of 207 fecal specimens collected from children with acute gastroenteritis between December 2001 and April 2003 in Yunnan Province, China were examined for the presence of adenoviruses, and group A and C rotaviruses, by RT-multiplex PCR. The detection rate of these three viruses was 55.1% (114 out of 207 specimens), among which adenovirus and group A and C rotaviruses were identified in 11, 101 and 1 fecal specimen, respectively. Furthermore, one specimen was found to be positive for co-infection with adenovirus and group A rotavirus. An epidemic of acute gastroenteritis was also identified as peaking mainly in October and November. Taken together, our results clearly indicate that this novel assay provides a potentially rapid and convenient tool for epidemiologic investigation of diarrhea caused by adenovirus and group A and C rotaviruses.