Primary thyroid epithelial cultures respond to cAMP agonists by stimulating both Cl- secretion and Na+ absorption 1. Single channel measurements indicate the presence of cAMP-gated Cl- channels and amiloride-sensitive Na+ channels in thyrocytes, suggesting that the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the epithelial Na+ channel (ENaC) mediate vectorial transport. Early studies localized CFTR to subapical compartments in thyroid 2, but formal demonstration of molecular expression has not been reported for either CFTR or ENaC, and whether CFTR mediates the thyroid Cl- secretory response remains unaddressed. To test this open question, we grew primary cultures of CFTR +/+ (wt) and CFTR -/- (ko) piglet thyroids (obtained from the Welsh lab, HHMI, U. Iowa) on permeable supports and assessed short-circuit current (Isc) in response to 1) the cAMP agonist, isoproterenol, and 2) amiloride. Serosal isoproterenol (10 µM) activated Isc in CFTR +/+, but not CFTR -/-, primary cultures (4.9 ± 1.67 μA/cm2 vs -0.42 ± 0.16 μA/cm2; mean ± sem; n=9 monolayers from 4 wt thyroids and 4 monolayers from 2 ko thyroids; p < 0.05 by unpaired t-test). Amiloride (10 μM, mucosal) inhibition of Isc was significantly greater in ko compared to wt monolayers (79 ± 4.4% vs 37 ± 7.4% of total resting Isc; n=5 ko and 7 wt; p < 0.05). To address if increased amiloride-sensitive Isc results from altered transcription of ENaC subunits, we performed quantitative, real-time reverse transcriptase polymerase chain reaction (qRT-PCR; described previously 3) using published primer pairs for α-, β- and γ-ENaC 4. Fold-changes in α-, β- and γ-ENaC mRNA levels from ko cultures did not differ significantly (ns) from wt: 1.28 ± 0.21 (α), 0.95 ± 0.11 (β) and 1.06 ± 0.15 (γ); n=5 pairs. Immunoblot quantification of ko vs wt cell protein lysates gave fold-changes in α-, β- and γ-ENaC subunit levels of 1.33 ± 0.1 (α; 95 kDa), 0.77 ± 0.13 (α; 32 kDa), 0.86 ± 0.16 (β; 90 kDa), 0.98 ± 0.26 (γ; 93 kDa) and 1.04 ± 0.29 (γ; 83 kDa) (mean ± sem; n=3 pairs each; p > 0.05 for all; ns). These findings indicate endogenous expression of CFTR and ENaC in thyroid and unequivocally show the essential role of CFTR in cAMP-mediated, bidirectional ion transport. Our data suggest that the functional relationship between CFTR and ENaC in the thyroid mirrors that in the airway surface epithelium. However, increased Na+ absorption appears not to result from altered expression of ENaC mRNA or protein subunits.