In previous work we demonstrated that inhibition of the cystic fibrosis transmembrane conductance regulator Cl^– channel (CFTR) retarded renal cyst growth (Li et al. 2004). To investigate the role of CFTR in cyst formation, we studied cyst formation by MDCK cells stably expressing wild-type human CFTR (WT-CFTR-MDCK) or loss-function mutation F508del (F508del-CFTR-MDCK) (Mendes et al. 2005). We also tested the effects of a specific CFTR blocker CFTR_inh-172 (Ma et al. 2002) on WT-CFTR-MDCK cyst formation. For cyst formation assays, we seeded MDCK cells in 24-well plates containing collagen gel and cultured them with media containing 10% FBS and forskolin (10 μM) for 6 days. To study the effects of CFTR_inh-172 on cyst formation, we treated WT-CFTR-MDCK cells from day 0 with the drug at 10 μM (Ma et al. 2002). On day 6, we counted all cysts with a diameter > 50 μm in each well. Using digital images, we measured area of individual cyst with ImageJ software. Assuming that cysts are spherical in shape, we calculated cyst volume (4/3 × π × r³). For cell proliferation assays, MDCK cells were seeded in 12-well plates containing MDCK media with 10% FBS and 10 μM forskolin in the absence or presence of CFTR_inh-172 (10 μM). In the presence of forskolin, WT-CFTR-MDCK cells formed more cysts (9.2 ± 0.8, n = 37 wells) with larger volume (0.23 ± 0.02 mm³, n = 265 cysts) compared to parental MDCK cells (2.0 ± 0.3, n = 24 and 0.10 ± 0.01 mm³, n = 48, P < 0.01, Student’s unpaired t-test). In contrast, F508del-CFTR-MDCK cells formed fewer cysts (0.4 ± 0.1, n = 27, P < 0.01 vs. parental MDCK cells) with smaller volume (0.02 ± 0.01 mm³, n = 11, P < 0.05 vs. parental MDCK cells). CFTR_inh-172 (10 μM) reduced significantly both the number and volume of cysts formed by WT-CFTR-MDCK cells (3.7 ± 0.8, n = 15 and 0.08 ± 0.01 mm³, n = 44, P < 0.01 vs. untreated WT-CFTR-MDCK). Cell proliferation and fluid secretion are important factors affecting renal cyst formation (Sullivan et al. 1998). To exclude the possibility that differences in cyst formation were caused by variation in the rate of cell growth, we studied the proliferation of the three MDCK cell lines with the same conditions used for cyst formation assays. At day 6, there was no significant difference in cell numbers among all the experimental groups (P > 0.05). We interpret our data to suggest that wild-type human CFTR promotes renal cyst formation mainly through its role of mediating Cl^– secretion and hence, water transport into the cyst lumen. Either a loss-function mutation or a CFTR inhibitor attenuates greatly renal cyst formation.