Primary cell cultures enriched in mucin-producing cells and basal cells were established from the trachea of the domestic fowl. Epithelial cells were selectively removed from the trachea after incubation in 0.1% pronase/0.1% EDTA in Moscona's saline. The majority of the ciliated cells were removed during the initial 30 minutes of incubation. After 50 minutes of incubation, aggregates of mucin-producing cells and basal cells were removed in large numbers. The cellular aggregates rapidly attached to a collagen-coated substratum and the cells spread out on the culture surface. The mucin-producing cells retained their AB/PAS-reactive secretory granules. The basal cells replicated and as the culture approached confluency, these cells developed a fine dusting of AB/PAS-reactive material; later, larger secretory granules appeared in the cells. These observations suggest that mucin-producing cells are capable of retaining their AB/PAS-reactive secretory products in primary culture and that basal cells are capable of differentiating into mucin-producing cells in vitro.