Eotaxin is an eosinophil-specific chemoattractant originally identified in bronchoalveolar lavage fluid after allergen challenge of sensitized guineapigs. We have determined and quantified for the first time the cellular sources of guinea-pig lung eotaxin and localized gene expression in structural cells of large and small airways and in alveolar macrophages.We used anti-guinea-pig eotaxin monoclonal and polyclonal antibodies and a complementary ribonucleic acid (cRNA) probe to detect eotaxin protein and cytoplasmic messenger ribonucleic acid (mRNA) transcripts by the techniques of immunohistochemistry and in situ hybridization in: 1) naive; 2) ovalbumin-sensitized/ saline-exposed; and 3) ovalbumin-sensitized/ovalbumin-challenged animals (n=5 for each group).Compared with the naive animals, there was a fivefold increase of eotaxin protein and a 25 fold upregulation of eotaxin gene expression in the airway epithelium 3 h after ovalbumin challenge of sensitized animals (p<0.001). The average percentages of alveolar macrophages staining for eotaxin protein and mRNA in the naive animals were approximately 30 and 10% respectively: both increased significantly in the sensitized/ovalbumin-challenged animals to 78 and 57%, respectively (p<0.0001). Compared with the naive animals, the procedure of sensitization and saline exposure significantly increased eotaxin gene expression in both bronchial epithelium and alveolar macrophages (p<0.01): the upregulation at these two sites showed a strong positive association (rcorr=0.95; p<0.0001).The results indicate that there are multiple cellular sources of guinea-pig lungderived eotaxin, including bronchial and bronchiolar epithelial cells, airway smooth muscle, bronchial vascular endothelium, and chondrocytes and alveolar macrophages, and that there are relatively rapid and marked increases of eotaxin protein and gene expression in airway epithelium and alveolar macrophages following allergen challenge.