Cross-species comparative studies have highlighted differences in the response of cystic fibrosis transmembrane conductance regulator (CFTR) homologues to small-molecule modulators (1, 2). Compared to human CFTR, ovine CFTR shows a high degree of sequence conservation (91% identity and 95% similarity at the amino acid level) (3), but exhibits enhanced conductance and ATP-dependent channel gating. Therefore, we were keen to characterise the pharmacology of ovine CFTR. In this study, we investigated the effects of the CFTR potentiator phloxine B (4) and the inhibitor glibenclamide (5) on the single-channel activity of ovine CFTR using excised inside-out membrane patches from transiently transfected Chinese hamster ovary K1 (CHO-K1) cells at 37 0C (1). In contrast to its potentiation of human CFTR, low micromolar concentrations of phloxine B (0.1 – 5 μM) were without effect or inhibited weakly ovine CFTR Cl- currents (e.g. phloxine B (1 μM), human, Idrug/Icontrol = 134 ± 11%, n = 9, P < 0.05; ovine, Idrug/Icontrol = 98 ± 4%, n = 11; P > 0.05; means ± SEM (n observations); Student’s t-test). However, elevated concentrations of phloxine B (≥ 10 μM) inhibited strongly both human and ovine CFTR (n = 4 – 8). Single-channel studies indicated that phloxine B (1 μM) potentiated human CFTR by increasing open probability (Po) (control, Po = 0.31 ± 0.03; phloxine B, Po = 0.54 ± 0.03; n = 15; P < 0.05). However, phloxine B had little impact on the Po of ovine CFTR (control, Po = 0.46 ± 0.04; phloxine B, Po = 0.46 ± 0.05; n = 8; P > 0.05). The open-channel blocker, glibenclamide (50 μM) inhibited ovine CFTR Cl- currents, albeit with reduced efficacy compared with human CFTR (human, Idrug/Icontrol = 31 ± 3%, n = 8; ovine, Idrug/Icontrol = 44 ± 2%, n = 9; P < 0.05). To investigate the mechanism of glibenclamide inhibition of ovine CFTR, we examined the voltage-dependence of channel block. Although glibenclamide inhibition of ovine CFTR was weaker than that of human CFTR (human, Kd(0 mV) = 16 ± 2 μM; ovine CFTR, Kd(0 mV) = 42 ± 12 μM, n = 5; P < 0.05), there was no difference in the electrical distance across the membrane sensed by glibenclamide (human CFTR, z’ = 0.25 ± 0.05, n = 4; ovine CFTR, z’ = 0.31 ± 0.05, n = 5; P = 0.44). We conclude that the pharmacological profile of ovine CFTR shows similarities to, but also differences from that of human CFTR. Variations in the pharmacology of ovine CFTR might result from subtle differences in the three-dimensional structure of CFTR and the local environment (e.g. hydrophobicity and charge) in the vicinity of drug-binding sites.