Fast regulation of the depth of the surface liquid layer (ASL) covering ciliated airway epithelium is critical for the function of the muco-ciliary clearance mechanism. Nucleotides (ATP and UTP) released to the surface layer are most likely involved in regulation of NaCl transport through interaction with apical purinergic receptors. The natural airway epithelium absorbs Na⁺ along a cellular pathway through apical ENaC and allows passive Cl^– absorption through the anion selective paracellular pathway. At the same time, a minor cellular Cl^– secretion takes place involving CFTR and calcium activated Cl^– channels. In order to evaluate the involvement of the different pathways in the regulation of NaCl transport, the effects of nucleotides on cellular Na+ absorption, cellular Cl^– secretion and paracellular diffusion were investigated. Under short-circuited conditions, tracer fluxes of Na⁺, Cl^– and mannitol were measured, in addition to electrophysiological transport parameters short circuit current (I_sc) and epithelial conductance (G_t), in rabbit nasal airway epithelia, from humanely killed animals, mounted in Ussing-chambers. Mucosal nucleotides (ATP or UTP, 200μM) inhibited amiloride-sensitive Na⁺ absorption (from 91.1±10.2 to 55.8±9.5 nmol min^-1 cm^-2; n=8; p<0.01) but only slightly increased Cl^– secretion (from 35.7±8.8 to 50.2±12.0 nmol min^-1 cm^-2; n=7; NS). From changes in G_t (UTP: -23.4±1.1%; n=101; p<0.001) it was calculated that nucleotides also caused a large decrease in paracellular conductance (G_s). Flux measurements showed that nucleotides decreased passive paracellular Cl^– fluxes (from 135.7±8.7 to 114.6±12.5 nmol min^-1 cm^-2; n=14; p<0.02) while passive Na⁺ fluxes and mannitol permeability were unaffected (Na⁺: from 35.1±4.5 to 37.5±4.2; n=8; NS. Mannitol: from 3.29±0.45 to 3.13±0.43 10^-6 cm s^-1; n=16; NS). The effects of nucleotides were mimicked by ionomycin (1μM), which also prevented the effects of nucleotides on I_sc and G_t. Stimulation of cAMP production with a purinergic P1 receptor agonist adenosine (200μM) or by forskolin (8μM) treatment had little effect on I_sc but increased G_t to an extent that indicated a substantial increase in G_s. In agreement it was found that forskolin increased passive paracellular Cl^– fluxes (from 126.4±19.6 to 173.6±18.2 nmol min^-1 cm^-2; n=6; p<0.002). The results suggest that nucleotides released to the ASL exert an autocrine regulatory function on airway epithelial ion transport, primarily by inhibiting net NaCl absorption, while stimulation of Cl^– secretion is of minor importance. An increase in [Ca²⁺]_i evoked by purinergic receptors probably mediates inhibition of ENaC channels and down-regulation of paracellular anion (Cl^–) permeability, while the opposite paracellular effect of cAMP suggests that this pathway is under dual control.