Multiple signaling mechanisms regulate epithelial cell tight junction (TJ) assembly and maintenance. Several G proteins are likely to regulate these processes, but only G(i/o) have been specifically tested. Treatment of MDCK cells with cholera toxin, a G alpha (s) activator, accelerated TJ development in the calcium switch as measured by the time to half-maximal [T-50 (H)] transepithelial resistance (TER). G alpha (s) was predominantly localized in the lateral membrane, but a fraction colocalizes with ZO-1 in the TJ. MDCK cell lines expressing epitope-tagged G alpha (s) and constitutively active (R201C alpha (8)) showed a similar localization. TJ assembly was significantly faster in R201C alpha (s)-MDCK cell lines (T-50 (H) of 1.7 versus 3.3 h for controls) without detectable differences in cAMP levels. Confocal studies showed R201C alpha (s)-MDCK cells more rapidly localized ZO-1 and occludin into the developing TJ without affecting E-cadherin or Na+/K+ ATPase localization. Endogenous G alpha (s) and R201C alpha (s) were immunoprecipitated with ZO-1 at baseline and during TJ assembly. The data supports a model of multiple G alpha subunits interacting with TJ proteins to regulate the assembly and maintenance of the TJ.