Voltage-gated K⁺ currents in rat somatotrophs are increased by somatostatin (SRIF) through unidentified G protein. In this experiment, somatotroph-enriched cells (up to 85%) were obtained from ovine pituitary glands and further identified by the increase in K⁺ currents by SRIF. The whole cell recording was employed to study the voltage-gated K⁺ currents. A reversible increase in K⁺ currents (up to 150% of control) was obtained in response to local application of SRIF (10 nM) but not vehicle. When the guanosine 5'-O-(3-thiotriphosphate) was included in the pipette solution (200 µM), the recovery phase of K⁺ current response to SRIF was abolished. Inclusion of guanosine 5'-O-(2-thiodiphosphate) (200 µM) in pipette solution blocked the K⁺ current response to SRIF. Intracellular dialysis of antibodies against _o-, _i-, _i-1-2-, or _i-3-subunits of G proteins via patch pipettes was confirmed by immunofluorescent staining of the antibodies. Antibody dialysis alone did not modify voltage-gated K⁺ currents. Dialysis of anti-_i or anti-_i-3 antibodies significantly attenuated the increase in K⁺ currents that was obtained after application of 10 or 100 nM SRIF. Dialysis with anti-_o, anti-_i-1-2, or heat-inactivated (60°C for 10 min) anti-_i antibodies did not diminish the effect of SRIF on K⁺ currents. We conclude that the G_i-3 protein mediates the effect of SRIF on voltage-gated K⁺ currents in ovine somatotrophs.