Ion channels underlying the resting membrane potential were examined in human fetal airway smooth muscle (ASM). Tissue was obtained from the Medical Research Council Tissue Bank, London, UK. ASM cells were enzymatically dispersed, and ion currents were examined using a patch clamp. Although all cells were of similar size and stained intensely for vimentin, only approximately 50% stained intensely for smooth muscle alpha-actin or myosin heavy chain. Depolarization induced a tetraethylammonium (TEA)- and charybdotoxin (ChTX)-sensitive outward current that varied widely among cells (<50 to >2000 pA at +100 mV), and a smaller nonselective cation current that was similar in all cells (approximately 20 pA at +100 mV). The TEA-sensitive current was associated with three types of large conductance, ChTX-sensitive K+ channel: a 200-pS channel, which was active at negative potentials and low [Ca2+], as described for freshly isolated adult ASM, and two other K+ channels of 100 and 150 pS, previously observed only in adult ASM proliferating in culture. ChTX, but not 4-aminopyridine, caused a substantial depolarization in the current clamp mode, suggesting that, in contrast to ASM from other species or vascular smooth muscle, large conductance K+ channels rather than a delayed rectifier are the major determinant of membrane potential in this tissue. Our results show a distinct similarity between fetal ASM and adult ASM proliferating in culture. We suggest that the heterogeneity in current density and staining reflect different degrees of differentiation, rather than different cell types, and that the 100- and 150-pS K+ channels are specifically associated with a proliferative phenotype in human ASM.