Although histamine is hypothesized to mediate symptoms induced by viral upper respiratory infections, elevations of this mediator have not been observed in nasal lavage fluids recovered from patients with viral upper respiratory infections. The purpose of this study was to use a novel method to determine whether histamine is released during experimental influenza A infection. Healthy adults (n = 15) were cloistered and inoculated intranasally with influenza A virus, and monitored for infection and illness. Daily morning void urines were collected and assayed for histamine and its metabolites by gas chromatography-mass spectrometry. Total histamine was calculated for each urine specimen by summing the assayed values of histamine and its metabolites. All subjects were infected and developed illness. ANOVA documented a significant effect of study day (viral infection) on urinary levels of total histamine (P < 0.02). Pairwise analysis showed a significant elevation 2 days after inoculation. These results provide the first direct evidence that histamine is released in vivo during infection with a virus that causes cold/flu symptoms.