Introduction: Human adenoviruses (HAdV) play an important role in the aetiology of severe acute lower respiratory infection, especially in immunocompromised individuals. The aim of the present study was to detect HAdV using two different methods, direct fluorescence assay (DFA) and nested polymerase chain reaction (nested PCR), in samples collected from patients with acute respiratory infection (ARI) within 7 days of symptom onset. Methods: Samples (n=643) were collected from patients in different risk groups from 2001 to 2010: 139 adult emergency room patients (ERP); 205 health care workers (HCW); 69 renal transplant outpatients (RTO); and 230 patients in a haematopoietic stem cell transplantation program (HSCT). Results: Adenovirus was detected in 13.2% of the 643 patients tested by DFA and/or PCR: 6/139 (4.3%) adults in the ERP group, 7/205 (3.4%) in the HCW group, 4/69 (5.8%) in the RTO group and 68/230 (29.5%) in the HSCT patient group. Nested PCR had a higher detection rate (10%) compared with the DFA test (3.8%) (p<0.001). HSCT patients exhibited a significantly higher rate of HAdV infection. Conclusions: The adenovirus detection rate of the nested PCR assay was higher than that of the DFA test. However, the use of molecular methods in routine diagnostic laboratory work should be evaluated based on the specific circumstances of individual health services.