The goals of our study were to isolate smooth muscle cells from the trachealis muscle of adult dogs and to characterize the cells morphologically when they were maintained in primary culture. Enzymatic digestion of the muscle yielded 4.8 +/- 1.8 X 10(6) viable smooth muscle cells per gram of tissue. When placed in culture, these cells rapidly proliferated until confluence was reached. The proliferating cells in culture differed from the cells in the intact tissue in that they stained less intensely for smooth muscle myosin, developed immunofluorescent staining for the intermediate filament protein vimentin, and lost many of the ultrastructural properties of the intact muscle. Only within nodules of cells in the confluent cultures were these ultrastructural properties preserved. Cultures of canine tracheal fibroblasts differed from these smooth muscle cell cultures in that the fibroblasts did not stain for smooth muscle myosin and did not form nodules at confluence. We concluded that adult canine airway smooth muscle cells may be maintained in primary culture, that the confluent cultures contain nodules of cells with many morphologic characteristics of the intact muscle, and that these preparations may be distinguished from cultured canine tracheal fibroblasts on specific morphologic grounds.