Epithelial cells from individuals with asthma or from allergen-sensitized mice contain intracellular interleukin (IL)-16 protein, not present in epithelial cells from individuals without asthma or unsensitized mice. IL-16 is only present in the bronchoalveolar lavage (BAL) fluid following airway challenge with either allergen or vasoactive amine. This suggests that the initial response to allergen (sensitization) results in synthesis but not secretion of IL-16. In this study, we investigated what factors produced during the sensitization phase are responsible for epithelial cell priming for IL-16 production. We determined that ovalbumin (OVA)-sensitized mice have an increase in systemic tumor necrosis factor-alpha levels, and that serum or BAL fluid stimulation of bronchial epithelial cells results in production of IL-16 that is subsequently secreted only following serotonin stimulation. The mechanism for IL-16 production was shown to be caspase-3-dependent, and serotonin-induced secretion of IL-16 required binding of the serotonin type 2 receptor. The relevance of the priming effect associated with sensitization for IL-16 production and storage was confirmed in vivo by serotonin airway challenge of OVA-sensitized mice, resulting in rapid secretion of IL-16 into BAL fluid. As IL-16 has been shown to regulate CD4+ cell recruitment and activation, and is detected early following airway challenge of individuals with asthma, this two-step process for IL-16 production by epithelial cells may represent a rapid response mechanism in the orchestration of allergic airway inflammation.