We evaluated the role of extracellular UTP and other nucleotides in the regulation of chloride (JCl) and fluid secretion (JV) across the pigmented rabbit conjunctiva. JV was determined in freshly excised conjunctival tissues mounted between two buffer reservoirs maintained in an enclosed environment at 37°C. Short circuit current (ISC) and 31Cl flux were measured using modified Ussing-type chambers. Fluid flux measurements were made with a pair of capacitance probes. After observing the baseline for 15 to 30 min, fluid flux was measured in the presence of mucosally applied nucleotides (10 μM) for a period of 30 min. Mucosal application of 10 μM each of UTP, UDP, ATP, ADP, AMP, adenosine, and ATP-γ-S transiently stimulated fluid secretion across the conjunctiva to a significant extent for 10 to 15 min. Other nucleotides did not show any significant effect. The stimulation of fluid secretion correlated well with the stimulation in ISC (r2 = 0.85). UTP (0.1-1000 μM) led to a maximal increase in fluid secretion by 11.72 ± 0.48 μl/(h · cm2) with an EC50 value of 10.39 ± 1.08 μM. ATP (0.1-1000 μM) caused a maximal increase in fluid secretion by 11.89 ± 0.88 μl/(h · cm2) with an EC50 value of 17.23 ± 2.63 μM. Adenovirus type 5 (Ad5) infection significantly decreased both net 36Cl secretion across the conjunctiva by ∼56% and the rate of fluid secretion by ∼56%. UTP (10 μM), but not 1 mM 8-bromo-cAMP, was able to elicit a normal stimulatory response in the Ad5-infected tissues. In conclusion, mucosal application of purinergic nucleotides may be therapeutically important in restoring ion and fluid secretion in the diseased conjunctiva.