Guinea pigs exposed to cotton dust (35 mg/m3 for 2 hrs) exhibited increases in the following pulmonary parameters: 1) CO₂-challenged breathing rate, 2) the number of ravaged leukocytes, and 3) the activity of alveolar macrophages. A correlation exists between the number of lavaged leukocytes and the breathing rate during CO₂ challenge. To investigate whether leukocytes are essential for these pulmonary responses, we determined the effect of leukocyte depletion on the responses of guinea pigs 18 hrs after cotton dust exposure (35 mg/m³ for 2 hrs). Intraperitoneal injection of hydroxyurea (30 µg/g body weight for 5 days)2 days prior to cotton dust exposure resulted in partial depletion (60-66%) of circulating leukocytes. Such treatment did not significantly affect the pulmonary responses to inhalation of cotton dust. In contrast, a single ip injection of cyclophosphamide (200 µg/g body weight) 3 days prior to cotton dust exposure resulted in complete depletion of circulating leukocytes. Complete leukocytopenia resulted in a substantial inhibition of both CO₂-challenged breathing rate and the activation of alveolar macrophages in response to cotton dust exposure. These data suggest that the acute response of guinea pigs to cotton dust is mediated by pulmonary leukocytes.