Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl^- channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a 4-amino acid PKA activation site, RGET₆₉₁ of hClC-2 was investigated using WT and mutant (AGET, RGEA and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, [cAMP]_i, EP₂ or EP₄ receptor agonist activity. All fatty acids (lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid and elaidic acid) caused significant rightward shifts in concentration-dependent Cl^- current activation (increasing EC₅₀s) with mutant compared to WT hClC-2 channels, without changing time- and voltage-dependence, I-V rectification or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA₆₅₅/RGEA₆₉₁) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]_i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells cobiprostone increased hClC-2 Cl^- currents without increasing [cAMP]_i, while PGE₂ and forskolin-IBMX increased both. Fatty acids were not agonists of EP₂ or EP₄ receptors. L-161,982, a supposed EP₄ selective inhibitor, had no effect on lubiprostone activated hClC-2 Cl^- currents, but significantly decreased T84 cell barrier function measured by TER and fluorescent dextran transepithelial movement. The present findings show that RGET₆₉₁ of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]_i and EP₂ or EP₄ receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.