The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl^– channel with complex regulation. In previous work, we demonstrated that intracellular pH (pH_i) modulates CFTR activity by altering channel gating (Chen et al. 2003). To understand the underlying mechanism, we studied CFTR channel gating at pH_i values from 5.8 to 8.8 using excised inside-out membrane patches from C127 cells expressing recombinant wild-type human CFTR that each contain only a single CFTR Cl^– channel. The pipette (external) solution contained 10 mM Cl^– at pH 7.3, and the bath (internal) solution contained 147 mM Cl^–, PKA (75 nM) and ATP (0.3 mM); voltage was –50 mV. Analyses of dwell time histograms demonstrated that channel gating was best described by one open state and two closed states at all pH_i values tested. Burst analyses demonstrated that pH_i decreased mean burst duration (MBD) as pH_i increased (e.g. pH_i 5.8, MBD = 357 ± 32 ms; pH_i 7.3, MBD = 133 ± 4 ms; pH_i 8.8, MBD = 93 ± 6 ms; n ≥ 4; p < 0.05). In contrast, pH_i had complex effects on interburst interval (e.g. pH_i 5.8, IBI = 515 ± 56 ms; pH_i 7.3, IBI = 155 ± 5 ms; pH_i 8.8, IBI = 144 ± 10 ms; n ≥ 4). To understand better how pH_i changes CFTR channel gating, we used QuB software and the C₁ ↔ C₂ ↔ O and C₁ ↔ O ↔ C₂ kinetic schemes (C₁, long closed state; C₂, brief closed state; O, open state) to model channel gating. In the C₁ ↔ C₂ ↔ O gating scheme, pH_i predominantly modulated transitions between the C₁ and C₂ states. In the C₁ ↔ O ↔ C₂ kinetic scheme, pH_i markedly altered transitions between the C₁ and O states, while acidic pH_i also altered transitions between the O and C₂ states. These data suggest that pH_i regulates MBD and IBI primarily by altering transitions between the first two states in both kinetic schemes. Because ATP binding and hydrolysis at two ATP binding sites controls channel gating, we assume that ATP molecules drive transitions between the different gating states C₁, C₂ and O. Thus, we derived the relationship between open probability (P_o) and [MgATP] using the C₁ ↔ C₂ ↔ O and C₁ ↔ O ↔ C₂ kinetic schemes. By examining whether the relationship between P_o and [MgATP] is well fitted by the Michaelis-Menten equation, our data demonstrate that if only one ATP molecule controls channel gating, C₁ is the site of interaction with ATP in both the kinetic schemes. However, if two ATP molecules control channel gating, only the C₁ ↔ O ↔ C₂ kinetic scheme describes the interaction of CFTR with ATP and ATP interacts with both closed states C₁ and C₂ in this kinetic scheme. Based on these data, we proposed a kinetic model with two ATP molecules interacting with CFTR to explain how pH_i modulates CFTR gating.