Eotaxin is the major eosinophil chemoattractant found in bronchoalveolar lavage (BAL) fluid from sensitized guinea pigs after antigen challenge. In this study we have performed immunostaining for eotaxin in airways obtained from challenged animals and examined purified guinea pig lung cells (epithelial cells > 98% purity, mast cells > 90% purity) for eotaxin mRNA and protein. In the airways of antigen (ovalbumin) challenged animals, significant amounts of epithelial cell eotaxin immunostaining were observed. Northern analysis of total RNA obtained from unchallenged, freshly isolated airway epithelial cells contained high levels of eotaxin mRNA. Semi-pure and high purity lung mast cell preparations (challenged or unchallenged) did not express eotaxin mRNA. Western analysis of supernatant fluids obtained from incubated airway epithelial cells demonstrated detectable amounts of eotaxin protein, with the majority of the protein being cell-associated. Thus, airway epithelial cells are identified as a major cellular source of eotaxin in the guinea pig pulmonary system.