In human bronchial epithelial cells (HBE) CFTR plays a critical role in mediating Cl- secretion. This important role is highlighted in cystic fibrosis, where CFTR mutations impact significantly leading to an increased risk of infection. Although CFTR is activated by a PKA dependent process, additional signalling pathways and a variety of accessory proteins also regulate its activity. In a previous patch clamp study pre-incubation with the tyrosine phosphatase inhibitor phenylarsine oxide (PAO) reduced the cAMP-activation of CFTR, suggesting that tyrosine phosphorylation is inhibitory (1). The aim of the current study was to determine whether this inhibitory action was also observed in epithelial monolayers. 16HBE14o− cells were grown on permeable supports until confluent and a transepithelial resistance of at least 200 Ωcm2 was attained. Inserts were mounted in an Ussing chamber with standard Krebs solution (basolateral side) and low Cl- Krebs (apical side, NaCl substituted with Na+ gluconate). Solutions were bubbled with 5% CO2, and all potential and resistance measurements corrected. At steady-state control measurements were taken for 5 minutes, before 10 µM forskolin (FSK) and 100 µM IBMX were added to activate CFTR. 10 µM CFTRinh172 was then added to the apical side of the insert to provide an indication of CFTR function. 10 µA of current was injected each minute to allow for the calculation of the equivalent short circuit current (ISC). In a second set of inserts cells were pre-incubated with 10 µM PAO before exposure to FSK and IBMX. Statistical significance was tested using ANOVAs and Student’s t-test as appropriate and assumed at the 5% level. In control inserts initial Vte was 6.40 ± 1.04 mV, and addition of FSK/IBMX increased this to 9.49 ± 1.81 mV. CFTRinh172 decreased Vte to 4.16 ± 0.97 mV (n=13). ISC increased from 19.7 ± 1.66 µS/cm2 to 24.8 ± 2.70 µS/cm2 with FSK/IBMX, and then fell to 11.1 ± 1.37 µS/cm2 with CFTRinh172. The CFTRinh172-sensitive Vte and ISC were 5.32 ± 1.12 mV and 13.8 ± 1.96 µS/cm2. In PAO incubated inserts the response to FSK/IBMX was absent. Vte was 5.54 ± 1.44 mV versus 3.68 ± 1.07 mV and ISC 18.5 ± 2.23 µS/cm2 versus 18.1 ± 2.09 µS/cm2 in the absence and presence of FSK/IBMX, respectively (n=9). CFTRinh172 gave a decrease in both Vte and ISC. However the magnitude of the response was attenuated compared to control. In PAO treated cells the CFTRinh172 -sensitive Vte was 1.80 ± 0.84 mV and ISC was 5.56 ± 2.01 µS/cm2. These data support the hypothesis that tyrosine phosphorylation has an inhibitory action on CFTR function and highlight the complex nature of the regulation of CFTR. Further work is now investigating whether this inhibitory action is mediated via a change in the activity of the channels or whether there is an impact on channel trafficking to or removal from the membrane.