Research scientists, laboratory technicians, and animal handlers who work with animals frequently report respiratory and skin symptoms from exposure to laboratory animals (LA). However, on the basis of prick skin tests or RASTs, only half are sensitized to LA. We hypothesized that aerosolized endotoxin from mouse work is responsible for symptoms in nonsensitized workers. We performed a cross-sectional study of 269/310 (87%) workers at a research institution. Subjects completed a questionnaire and underwent prick skin tests (n = 254) or RASTs (n = 16) for environmental and LA allergens. We measured airborne mouse allergen and endotoxin in the animal facility and in research laboratories. Of 212 workers not sensitized to mice, 34 (16%) reported symptoms compared with 26 (46%) of mouse-sensitized workers (p < 0.001). Symptomatic workers were more likely to be atopic, regardless of mouse sensitization status. Symptomatic non-mouse-sensitized workers spent more time performing animal experiments in the animal facility (p = 0.0001) and in their own laboratories (p < 0.0001) and had higher daily endotoxin exposure (p = 0.008) compared with asymptomatic coworkers. In a multivariate model, daily endotoxin exposure most strongly predicted symptoms to mice in non-mouse-sensitized workers (odds ratio = 30.8, p = 0.003). We conclude that airborne endotoxin is associated with respiratory symptoms to mice in non-mouse-sensitized scientists and technicians.