Cystic fibrosis is caused by genetic mutations that lead, via a variety of molecular mechanisms, to loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Opening of CFTR channels is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the channel pore are not known. To gain some information on conformational changes in the pore, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine reactive reagents to cysteines introduced into the pore-forming sixth transmembrane region (R334-P355) of a cysteine-less variant of CFTR. Our results show that several of the cysteine substitutions themselves (R334C, K335C, I336C, T338C, S341C, F342C, R347C, T351C, R352C, Q353C, F354C) led to quantifiable changes in current-voltage relationship shape, suggesting that mutagenesis of these residues has some impact on pore function. Moreover, we find that methanethiosulphonate (MTS) reagents modify irreversibly cysteines substituted for (from outside to inside) F337, T338, S341, I334, V345, M348, A349, R352 and Q353 when applied to the cytoplasmic side of open channels, leading to decreases or increases in macroscopic current amplitude. In contrast, external MTS reagents modified T338C and S341C but not I344C, V345C or M348C, confirming that MTS reagents are not permeant in CFTR. When used to pretreat excised membrane patches, we find that the pattern of modification by internal [2-sulphonatoethyl] methanethiosulphonate (MTSES) depends on the activation state of the channels. Thus, while cysteines located relatively superficially into the pore (V345C, M348C) can be modified in both activated and non-activated channels, those more deeply into the pore (T338C, S341C, I344C) can only be modified after the channels have been activated by protein kinase A and ATP. Access of a permeant anion, Au(CN)2-, from the cytoplasmic solution to the central region of the pore was similarly regulated by channel activation state. The pattern of MTS modification we observe allows us to divide the pore into distinct “regions” that we refer to as the outer vestibule, narrow region and inner vestibule. Prior to channel activation, internally applied MTS reagents are restricted to the inner vestibule, whereas following activation they are able to penetrate further into the pore, reaching in to the narrow region. We therefore propose that channel activation involves removal of a restriction preventing access from the cytoplasm to the pore narrow region, and suggest that this mechanism involves opening of a channel “gate” near the outer end of the inner vestibule.