The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel contains two cytoplasmic nucleotide-binding domains (NBDs). After phosphorylation of the R domain, ATP interacts with the NBDs to regulate channel activity. To learn how the NBDs regulate channel function, we used the patch-clamp technique to study CFTR and variants which contained site-directed mutations in the conserved Walker A motif lysine residues in either NBD1 (K464A), NBD2 (K1250A and K1250M), or both NBDs simultaneously (K464A/K1250A). Studies in related proteins suggest that such mutations slow the rate of ATP hydrolysis. These mutations did not alter the conductive properties of the channel or the requirement for phosphorylation and ATP to open the channel. However, all mutations decreased open state probability. Mutations in NBD1 decreased the frequency of bursts of activity, whereas mutations in NBD2 and mutations in both NBDs simultaneously prolonged bursts of activity, as well as decreased the frequency of bursts. These results could not be attributed to altered binding of nucleotide because none of the mutants studied had reduced 8-N3ATP binding. These data suggest that the two NBDs have distinct functions in channel gating; ATP hydrolysis at NBD1 initiates a burst of activity, and hydrolysis at NBD2 terminates a burst.