Background: The state of Rio de Janeiro has been important since 1986 as a portal for the introduction of dengue virus (DENV) into Brazil and dissemination of the virus throughout the country. This study describes an active surveillance of DENV in the state of Rio de Janeiro from 2004 to 2008. Method: A total of 14 408 samples from patients suspected to be infected with DENV were tested by virus isolation, and nested reverse transcriptase (RT)-PCR assay or anti-DENV dengue IgM antibody capture ELISA (MAC-ELISA), or both. Results: By the use of these different methods, a total of 2324 (16.1%) cases were confirmed as dengue infection. The study covers an inter-epidemic period (2004–2005), the DENV-3 circulation in 2006, the re-emergence of DENV-2 in 2007 and the severe epidemic caused by DENV-2 in the summer of 2008. During the period, 69 dengue fatal cases were reported, 14 (20.2%) deaths being attributable to DENV-3 and 55 (79.7%) to DENV-2. Conclusion: Our results emphasize the role of the laboratory in the early detection of dengue virus transmission and provide information on the dynamics of DENV introduction and spread, important for the assessment of intervention strategies.