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This study presents the results obtained in the monitoring of dengue virus (DENV) transmission in the Greater Metropolitan Region of the State of Rio de Janeiro, in the period 2000–2001. A total of 5324 serum samples from suspected cases of dengue were analysed in order to confirm dengue infection. The introduction of DENV-3 to the region in December 2000 resulted in the co-circulation of three serotypes: DENV-1, DENV-2 and DENV-3. In this study, virus isolation and/or reverse transcriptase PCR (RT–PCR) confirmed 52.3% (42/79) of DENV-3 cases, showing the importance of acute serum samples in the virological surveillance of the disease. Despite the introduction of a new serotype, an outbreak due to DENV-1 was observed in the municipality of Niteroi. The restriction site-specific PCR (RSS–PCR) patterns obtained for DENV-1 and DENV-2 isolated in that period showed that those strains belonged to the subtypes previously circulating in the state. DENV-3 RSS–PCR patterns confirmed that these viruses belonged to subtype C (Sri Lanka/India strains), represented by the strain circulating on the American continent. These data showed the importance of an active surveillance programme in countries where dengue is endemic. [Copyright &y& Elsevier]