Organic dust exposure in agricultural environments results in an inflammatory response that attenuates over time, but repetitive exposures can result in chronic respiratory disease. Animal models to study these mechanisms are limited. This study investigated the effects of single versus repetitive dust-induced airway inflammation in mice by intranasal exposure method. Mice were exposed to swine facility dust extract (DE) or saline once and once daily for 1 and 2 weeks. Dust exposure resulted in increased bronchoalveolar lavage fluid neutrophils and macrophages after single and repetitive exposures. Lavage fluid TNF-, IL-6, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)-2 were significantly increased after single and repetitive dust exposures, but were dampened in 2wk dust-exposed mice as compared to single exposure. Dust exposure induced PKC- and - activation in isolated tracheal epithelial cells, but were dampened with repetitive exposures. Ex vivo stimulation of alveolar macrophages from 2-week animals demonstrated reduced cytokine responsiveness and phagocytic ability. Significant lung pathology occurred with development of mixed mononuclear cellular aggregates (T- and B-lymphocytes, phagocytes) after repetitive dust exposure, a novel observation. Airway hyper-responsiveness to methacholine occurred after single dust exposure, but resolved after 2 weeks. Collectively, intranasal exposure to DE results in significant lung inflammatory and pathologic responses marked by a modulated innate immune response to single and repetitive dust exposures that is associated with PKC activity.