Dengue is the most important arthropod-borne viral disease, and it is a major public health problem in subtropical and tropical regions. The virus is transmitted to humans by the bite of infected female mosquitoes of the genus Aedes. The global resurgence of dengue is thought to be due to failure to control the Aedes populations, uncontrolled urbanization, population growth, climate change, and increased airplane travel. In this paper we describe the methods used to detect dengue virus infection in a patient who presented to a hospital in New York State. The patient was a 21-year-old female who had recently traveled from Thailand. Serologic testing, viral culture, and molecular methods, including reverse transcription-PCR (RT-PCR) and real-time RT-PCR, were tools used for diagnosis. Enzyme-linked immunosorbent assay and immunofluorescence assay of serum specimens indicated flavivirus infection. Conventional RT-PCR and sequencing, in addition to real-time RT-PCR of serum samples and nasal and throat swabs from the patient, confirmed dengue virus 1 (DEN-1) infection. A cytopathic effect was observed in virus cultures of the acute-phase serum samples and nasal swabs. DEN-1 was subsequently detected by RT-PCR from cell culture supernatants and by direct fluorescent-antibody assay staining of the cell culture monolayer. We show that a multipronged approach to the laboratory diagnosis of dengue infections can be used to successfully diagnose and differentiate the dengue virus serotypes. In addition, we show that both dengue viral RNA and infectious virus can be detected in respiratory specimens from an infected patient.