Purines regulate intraocular pressure. Adenosine activates Cl− channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl− channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion. The actions of nucleotides alone on Cl− channel activity of bovine PE cells were studied by electronic cell sorting, patch clamping, and luciferin/luciferase ATP assay. Cl−channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 μM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2, and cAMP activated Cl− channels without enhancing effects of 100 μM ATP. Buffering intracellular Ca2+activity with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid or blocking PGE2 formation with indomethacin inhibited ATP-triggered channel activation. The Rp stereoisomer of 8-bromoadenosine 3′,5′-cyclic monophosphothioate inhibited protein kinase A activity but mimicked 8-bromoadenosine 3′,5′-cyclic monophosphate. We conclude that nucleotides can act at >1 P2Y receptor to trigger a sequential cascade involving Ca2+, PGE2, and cAMP. cAMP acts directly on Cl−channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.