Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures on filters, an acute transient increase (phase I response; triggered by P2Y₂ receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability (phase II response). Phase II response is mediated by augmented calcium influx and protein kinase C-dependent increase in tight junctional resistance. The objective of the study was to determine the role of P2X₄ receptor as a mediator of phase II response. Human cervical epithelial cells express P2X₄ receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms by Northern blot analysis) and P2X₄ protein. Depletion of vitamin A reversibly downregulated P2X₄ receptor mRNA and protein and ATP-induced calcium influx. Depletion of vitamin A abrogated phase II response, and the effect could be partially reversed only with retinoic acid receptor (RAR)-selective retinoids but not retinoid X receptor (RXR) agonists. Depletion of vitamin A also abrogated protein kinase C increase in tight junctional resistance, and the effect could not be reversed with retinoids. Depletion of vitamin A also abrogated phase I increase in permeability and reversibly downregulated P2Y₂ receptor mRNA and ATP-induced calcium mobilization. However, in contrast to phase II response, both RAR and RXR agonists could fully reverse those effects. These results suggest that phase II response is mediated by a P2X₄ receptor mechanism.