The mechanisms governing maintenance of quiescence during pregnancy are unknown. This study characterizes a stretch-activated, TEA-insensitive K⁺ current found in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that the K⁺ currents described can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until term. The results of this study demonstrate that in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF using arachidonic acid or application of sodium bicarbonate causing intracellular acidification, respectively. Similarly, outward currents were inhibited following the application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by arachidonic acid, and 73.9 ± 4.2% after activation using sodium bicarbonate. In HEK293 cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF using arachidonic acid or sodium bicarbonate, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by arachidonic acid and 91.6 ± 3.4% after sodium bicarbonate activation. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with interfering RNA (siRNA) or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.