Thyrotropin releasing hormone (TRH) and high extracellular K+ induce prolactin release from rat pituitary cells (GH3 cells) in culture. TRH, as well as high K+ (50 mM), was shown to increase 45Ca2+ efflux from these cells. The effects of TRH and high K+ on membrane polarization was determined indirectly using the lipophilic cation, triphenylmethyl phosphonium ion (TPMP+). The resting membrane potential of GH3 cells was -50 +/- 1.7 mV. TRH, which induced a marked increase in 45Ca2+ efflux and stimulated prolactin release, had no effect on membrane polarization measured simultaneously. High K+, which also increased 45Ca2+ efflux and prolactin release, depolarized the GH3 cell as evidenced by a marked increase in [3H]TPMP+ efflux. These results suggest that TRH and high K+ may act via different mechanisms to increase intracellular Ca2+ and stimulate prolactin release.