Virus isolation is the most reliable evidence of infection. In the present study, we isolated virus from serum samples collected from confirmed dengue cases. When data were analyzed based on disease days, dengue viruses were isolated from 28 of 32 serum samples collected on disease day 5 or earlier. When analyzed based on fever days, dengue viruses were isolated from all the serum samples collected on fever day -3 or earlier, and from 10 of 13 samples collected on fever days -2 and -1. Viruses were isolated from one each of the serum samples collected on fever days 0, 1, 2 and 3, respectively. Virus was not, however, isolated from those collected on fever day 4 or later. The results of virus isolation and reverse transcriptase-polymerase chain reaction were consistent in 78 of 82 serum samples. These results suggest that virus isolation is a useful and sensitive technique for confirmation of dengue virus infection, especially when serum samples are collected before fever subsides.