Cystic fibrosis (CF) is a life-shortening genetic disorder resulting from loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1). To understand the pathophysiology of CF and test new therapeutics, large animal models have been developed using pigs (2) and sheep (3). Because CFTR function and pharmacology vary across species (4), the aim of this study was to investigate the single-channel behaviour of pig CFTR with that of human and sheep CFTR (5). Using the patch-clamp technique, we studied CFTR Cl- channels in excised inside-out membrane patches from CHO-K1 cells transiently transfected with CFTR constructs using a large Cl- concentration gradient ([Cl-]int, 147 mM; [Cl-]ext, 10 mM) (5). Like other CFTR orthologues, pig CFTR formed Cl- selective channels regulated by protein kinase A-dependent phosphorylation and intracellular ATP. However, when compared with human CFTR, pig and sheep CFTR differed in two important ways. First, the single-channel conductance of pig CFTR (9.7 ± 0.3 pS; n = 6) and sheep (9.9 ± 0.1 pS; n = 13) were greater than that of human CFTR (9.2 ± 0.1 pS; n = 5) (means ± SEM). Second, distinct differences were observed in the frequency and duration of channel openings. At 1 mM ATP, the long closures separating channel openings of pig CFTR (n = 6) were similar to those of human CFTR (n = 9), while those of sheep CFTR (n = 13) were 56% shorter. Moreover, the duration of channel openings of pig CFTR (414%; n = 6) and sheep CFTR (174%; n = 13) were noticeably longer than those of human CFTR (n = 9). As a result, the open probability (Po) of pig CFTR (0.68 ± 0.04; n = 12) and sheep CFTR (0.60 ± 0.02; n = 24) were greater than that of human CFTR (0.40 ± 0.02; n = 19). To explore how ATP gates CFTR, we examined the ATP dependence of Po between 0.03 and 3 mM ATP. By fitting mean Po data with the Michaelis-Menten functions, we determined the apparent affinity and efficacy of CFTR for ATP. When compared to human CFTR (KD = 230 µM, Po(max) = 0.66, r2 = 0.95; n = 4 – 16), ATP regulated pig CFTR (KD = 32 µM, Po(max) = 0.81, r2 = 0.82; n = 4) and sheep CFTR (KD = 77 µM, Po(max) = 0.70, r2 = 0.93; n = 5 – 13) with increased affinity and efficacy. Collectively, we conclude that: i) CFTR activity varies between species, decreasing in the rank order pig > sheep > human and ii) differences in current flow through open channels and ATP-dependent channel gating explain the variation in channel activity. In addition to assisting studies of CF animal models, these data provide insight into species-specific differences that will inform future analyses of CFTR structure and function with the potential to identify new CF therapeutics.