OBJECTIVE: To determine the level of genetic variation of human parainfluenza virus type 3 (HPIV-3), and to describe infection and co-infection characteristics of HPIV-3 in children. METHODS: Single respiratory samples from 856 pediatric patients with acute respiratory tract infection (ARI) in Hangzhou were collected from December 2009 to March 2013. All samples were screened for HPIV-3 by real-time RT-PCR and followed by HN sequencing and phylogenetic analysis. In all RSV positive specimens, we screened for the other pathogens, and co-infection characteristics were evaluated. RESULTS: A total of 9.6% of 856 samples were positive for HPIV-3, the nucleotide among the strains ranged from 96.9% to 100%. All Hangzhou strains were placed in C3 subgroup based on HN gene analysis. 49% (n=41) of all HPIV-3-positive children with ARI were found to be co-infected with at least one of the other pathogen. The highest co-infection rate of HPIV-3 was with HRV (n=17). Children in the younger groups (</=12 months old) were significantly more prone to be co-infected with other pathogen (chi(2)=4.78, P=0.029). Pneumonia infection rate was significantly higher in the mono-infection group than the co-infection group (chi(2)=3.92, P=0.048). CONCLUSION: HPIV-3 was an important pathogen in children with ARI in Hangzhou. HN gene variation rate was low, but showed a more local pattern. The co-infections with other respiratory viruses were popular. Except for pneumonia, no significant differences in other clinical presentation between the HPIV-3 mono-infection and co-infection groups were observed.