Extracellular ATP changes the transepithelial electrical conductance (GT) across cultures of human cervical cells acutely, in a biphasic manner that is characterized by a rapid increase (phase I) followed by a sustained decrease in GT (phase II). We tested the hypothesis that the phase II response is mediated by decreases in the permeability of tight junctions. We studied the effect of ATP on the relative mobilities of Cl- vs. Na+ (uCl/uNa) as calculated from changes in the dilution potential (Vdil). Vdil was induced by lowering NaCl from 130 to 10 mM in either the luminal or subluminal solutions bathing filters containing cells. uCl/uNa was 1.27 across cervical cultures and 1.34 across blank filters, compared with a level of 1.52 in free solution. Increases in GT induced by transepithelial hydrostatic or hypertonic gradients (which increase permeability of lateral intercellular space) had no effect on uCl/uNa. Increases in GT induced by lowering extracellular Ca2+ to < 0.1 mM increased uCl/uNa to levels obtained in blank filters, indicating abrogation of tight-junctional resistance. Phase I response and ionomycin (which produces a sustained phase I-like increase in GT) had no effect on uCl/uNa. The phase II response, however, decreased uCl/uNa from 1.27 to 1.24, and the effect could be abrogated by lowering extracellular Ca2+. These results indicate that phase II decreases in GT across cultured human cervical epithelium are mediated by acute decreases in tight-junctional permeability.