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1. Simultaneous bidirectional tracer fluxes of 22Na and 36Cl were measured under short-circuited conditions in isolated stripped segments of Amphiuma duodenum incubated in media containing Cl- and HCO3-. 2. Theophylline (10 mM) increased the short-circuit current (Isc), eliminated net Cl- absorption and elevated the residual flux by more than 50%. 3. Subsequent addition to acetazolamide lowered the Isc and reduced the residual flux. 4. Using double-barreled Cl- specific micro-electrodes the intracellular activity of Cl- averaged 27.1 mM in the presence of theophylline, consistent with active accumulation of Cl-. 5. Theophylline depolarized the mucosal membrane of villus epithelial cells by an average of 6.9 mV. 6. The findings indicate that the inhibition of Cl- absorption by theophylline does not result from complete inhibition of mucosal Cl- uptake and provide evidence that HCO3- secretion is enhanced by theophylline.