Primary tissue culture system from the middle ear and the tracheal epithelium of the guinea pig was established using collagen gel method. The cultured epithelium was studied by phase-contrast microscopy, light and scanning electron microscopy. The outgrowth area of the epithelium was quantitatively measured for period of up to ten days. In the middle ear cavity, the mucosal explants were sampled from various sites in order to compare their differential and proliferative activities. The mucosal explants attached on collagen substrates were composed of ciliated, non-ciliated, goblet and basal cells. This basic structure was similar to the natural middle ear epithelium. The ciliated cells showed well organized cilia. Most of the outgrowth cells devoid of fibroblastic cells in the monolayer were polygonal shaped with numerous microvilli. The morphology of the outgrowth cells changed from columnar or cuboidal to squamous shapes in the area away from the explants. There was a correlation between the distribution of the ciliated cells in the outgrowths and these in the explants. The explants with columnar or cuboidal ciliated epithelia sampled from the opening of the eustachian tube or its neighborhood formed more than eightfold outgrowth sheets in vitro. This is comparable to tracheal epithelia. On the other hand, the explants with simple squamous epithelia sampled from the area distal to the eustachian tube showed about fourfold proliferative activity. We concluded that this culture system would be useful for the study of cellular multiplication and differentiation mechanisms of the respiratory tract epithelium.