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University Department of Pharmacology, South Parks Road, Oxford OX1 3QT, U.K.

1. Cl-sensitive micro-electrodes were used to measure the intracellular Cl activity (a_Clⁱ) in smooth muscle cells of the guinea-pig vas deferens. The values obtained were compared with those of intracellular Cl (Cl_i) found by both ion analysis and ³⁶Cl efflux.

2. Various combinations of filling solution for recording membrane potential (E_m), and type of micro-electrode were tested. The most successful, which allowed continuous recording of a_Clⁱ for several hours, was a double-barrelled electrode using the reference liquid ion exchanger (RLIE; Thomas & Cohen, 1981). However, a_Clⁱ measured both by simultaneous impalements of separate cells with Cl-sensitive and conventional micro-electrodes, and by double-barrelled micro-electrodes, was about 42 mM in normal Krebs solution. This is five times higher than the value from a passive distribution. E_Cl was about -24 mV, more than 40 mV positive to E_m.

3. On complete removal of extracellular Cl (Cl_o), a_Clⁱ fell to an apparent level of about 3 mM. If this represents interference from other anions, the maximum error in E_Cl measured in normal Krebs solution is 2·5 mV. Replacement of Cl_o caused a rapid increase in a_Clⁱ. This must be caused by an active transport of Cl^- ions into the cell against their electrochemical gradient.

4. The stabilized values of a_Clⁱ measured at different levels of Cl_o agree surprisingly well with a_Clⁱ estimated from ion analysis and ³⁶Cl efflux, assuming that the intracellular activity coefficient was the same as measured in the normal Krebs solution. The relationship of a_Clⁱ to Cl_o was hyperbolic.

5. It is concluded that Cl-sensitive micro-electrodes accurately measure a_Clⁱ in smooth muscle cells. The remarkable agreement between the direct and indirect methods of measuring Cl_i suggests that Cl^- ions are not bound to a significant extent and that the compartment seen by the micro-electrodes is probably representative of the whole cell.

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