Culture of dispersed airway smooth muscle (ASM) cells with fetal bovine serum (FBS) induces rapid growth and modulation from a contractile to a synthetic phenotype, but this may be an artificial situation due to loss of tissue architecture. In this study, structural, functional and biochemical changes of ASM were examined in human intact bronchiole ring segments (200 to 600 micro m internal diameter) after organ culture for up to 6 days in 10% FBS or in D-STIM, an FBS-free medium formulated to maintain a contractile phenotype. ASM content was unchanged after culture for 3 or 6 days with D-STIM or FBS compared with fresh tissues. However, by 6 days culture with FBS reduced the maximum developed contraction to several agonists (carbachol, histamine, and KCl). Smooth muscle (sm)-alpha-actin and sm-myosin heavy chain (MHC) isoform 1 expression was similar for all culture conditions, though FBS reduced calponin and metavinculin content. Nonmuscle (nm) proteins, including total vinculin, beta-actin, and nm-MHC, were unchanged. Thus, ASM in human intact bronchioles maintains its functional, biochemical, and morphometric properties after culture in FBS-free (D-STIM) medium for at least 6 days, and for 3 days when cultured with FBS. These findings in organ culture may reflect more closely the in vivo situation in which tissue architecture is better preserved over cell culture, and may provide a basis for examining long-term effects of trophic or contractile stimuli on intact ASM in vitro that contribute to airway hyperresponsiveness and remodeling.