Background Clinical, virologic, and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. Methods US subjects with suspected ZIKV infection were enrolled. Clinical data and specimens were prospectively collected for ZIKV RNA detection and serologic and cellular assays. Confirmed ZIKV infection (cases) and ZIKV-negative (controls) subjects were compared. Dengue-experienced and dengue-naïve cases were also compared. Results We enrolled 45 cases and 14 controls. Commonly reported symptoms among cases and controls were maculopapular rash (97.8% and 81.8%), fatigue (86.7% and 81.8%), and arthralgia (82.2% and 54.5%), respectively. The sensitivity (94%) and duration of infection detection (80% positivity at 65–79 days after disease onset) by polymerase chain reaction were highest in whole-blood specimens. ZIKV-neutralizing antibodies had a half-life of 105 days and were significantly higher in dengue virus–experienced cases than naïve ones (P = .046). In intracellular cytokine staining assays, the ZIKV proteins targeted most often by peripheral blood mononuclear cells from cases were structural proteins C and E for CD4+ T cells and nonstructural proteins NS3, NS5, and NS4B for CD8+ T cells. Conclusions ZIKV RNA detection was more frequent and prolonged in whole-blood specimens. Immunoglobulin G (IgG) and neutralizing antibodies, but not IgM, were influenced by prior dengue infection. Robust cellular responses to E and nonstructural proteins have potential vaccine development implications.