Matrix metalloproteinases (MMPs) are involved in the remodeling of extracellular matrix, but are also proposed to have other roles. A previous report indicated that recombinant MMP2 protein decreased, while the non-specific MMP inhibitor, 1,10-phenanthroline, and an anti-MMP2 antibody increased CFTR chloride current [1]. Furthermore, a link between MMP activity and potassium ion channel regulation has been proposed that involves heparin-binding EGF-like growth factor shedding by MMP cleavage [2]. Here we investigate the possible regulatory role of MMPs and EGF on CFTR chloride current in Calu-3 cells. Calu-3 cells were grown on permeable supports until confluent at which point the apical fluid was removed producing an air-liquid interface (ALI). Supports were incorporated into an Ussing chamber 7-15 days after ALI and short circuit current (Isc) across the monolayer was measured. All filters were treated with 10µM amiloride before each experiment to block epithelial sodium channels. Initially, addition of 10µM forskolin to the basolateral side produced an increase in Isc of 7.2±4.1µA/cm2 (n=3), which was inhibited by 50M GlyH-101 by 99.6% (n=3). Acute addition of 5ng/ml EGF to the apical side of the monolayer resulted in a biphasic response. There was an initial transient increase (peak) in Isc of 2.7±1.8µA/cm2 (n=2), followed by a reduced but greater than baseline plateau phase (increase in Isc of 1.5±0.8µA/cm2;n=2). Increasing the EGF concentration to 50ng/ml resulted in no further significant increase in Isc. Apically applied 50µM GlyH-101 inhibited by 257% (n=2) the increase in Isc due to 5ng/ml EGF. Pre-incubation of Calu-3 cells with the EGF receptor inhibitor, AG1478, followed by stimulation with 5ng/ml EGF on the apical side resulted in complete inhibition of the EGF stimulated Isc. When the basolateral membrane was permeablised using 0.36mg/ml nystatin and a basolateral to apical chloride gradient applied, 5ng/ml EGF added to the apical side increased the Isc by 11.0 ± 0.3µA/cm2 (n=3, p<0.001). To investigate whether the gelatinases MMPs-2 and -9 were secreted by Calu-3 cells we used gelatin zymography to assay for MMP production. Unstimulated Calu-3 cells have very little detectable MMP activity in conditioned media. Addition of 5ng/ml EGF for 24 hours to conditioned media induced MMP-2 and MMP-9 production in Calu-3 cells. This activity could be inhibited by the general MMP inhibitor, GM6001. In conclusion, the data suggest that EGF stimulates MMP production. EGF, working via the EGFR, also stimulates Isc by activating CFTR chloride channels. Further work will be required to determine the regulatory link between the MMP activity and the CFTR channel activation.