The luminal surface of the airways is covered with the airway surface liquid (ASL), which plays an important role in host pulmonary defence. To fulfil this role its volume and composition must be controlled, and this is achieved through the regulation of ion, and hence liquid, transport across the epithelium. At rest, the airway epithelium is primarily absorptive. Na⁺ is absorbed from the ASL through apical Na⁺ channels (ENaC), and pumped across the basolateral membrane by Na⁺/K⁺ATPase. To maintain the electrochemical driving force for this process, basolateral K⁺ conductances (G_K) enable K⁺ efflux. Whilst a great deal is known about the control of ENaC, relatively little is known about the nature and regulation of G_K. The aim of this study was to investigate the basolateral G_K present in the human bronchiolar cell line, H441. H441 cells were grown to confluence on permeable supports (2) and mounted in Ussing chambers containing HCO₃^–-buffered Kreb’s solution. Transepithelial PD was monitored (voltage clamp; WPI) and recorded directly to computer using a Powerlab interface (ADInstruments). Once PD was stable (20min), the bathing solutions were replaced with solutions modified to impose an apical (135mM K⁺) to basolateral (4.7mM K⁺) K⁺ gradient and a maximal dose of amphotericin B was added (200µM). PD rose to a new, stable level (7.3±0.3 to 12.2±0.5mV, n=50) and then monolayers were short circuited. Ba²⁺ (5mM), clofilium (100µM) and clotrimazole (100µM) inhibited 52±10%, 76±15% and -6±4% of amphotericinB-induced I_K, respectively. 1-Ethyl-2-benzimidazolinone (EBIO, 1mM), an activator of Ca²⁺-activated K⁺ channels (1) increased I_K by 20.2±3.6µA cm^-2 (n=8). However, this was unaffected by a blocker of EBIO-sensitive K_Ca3.1 channels, clotrimazole. Unexpectedly, thapsigargin (1µM) had no effect on I_K. ATP (100µM) evoked a transient increase (21.0±1.9µA cm^-2, n=9) that was unaffected by either clofilium, Ba²⁺ or clotrimazole but inhibited by clamping PD at the K⁺ equilibrium potential (E_K, -89mV). Both adenosine (200µM) and forskolin (10µM) evoked small increases in I_K (3.4±2.0, n=6 and 5.4±1.6, n=4, respectively). H441 cells possess basolateral G_K that are regulated by a number of agonists known to control transepithelial Na⁺ transport, and include both those that alter intracellular Ca²⁺ concentration (ATP), and those controlling cAMP levels (forskolin and adenosine). The lack of a response to thapsigargin may indicate that G_K is regulated by transient increases in intracellular Ca²⁺ as evoked by ATP, rather than the bulk changes in cytoplasmic Ca⁺ levels induced by thapsigargin. Alternatively, ATP may exert its effect by some other, as yet unknown, action. Since clotrimazole had no effect on either basal I_K or following stimulation with EBIO, K_Ca3.1 channels are unlikely to underlie basolateral G_K in these cells.