Large conductance Ca2+-activated K+ channels play a critical role in regulating myometrium contractility. Their current density, mRNA, and total protein are greatly diminished in myometrium of late pregnant rats versus nonpregnant animals. Opposite to rats, in mice, channel mRNA and total protein increase in late pregnancy, but current density decreases as in rats. Here, we investigated the mechanism of these differences. Real time PCR and Western blots demonstrate that, in late pregnancy, channel transcript quantities and total protein were diminished in rats but up-regulated in mice. High resolution confocal microscopy of single myocytes showed that, in nonpregnant mice, channels were expressed in clusters at the surface membrane. In late pregnancy, although there was an overall increase in channel protein, its majority was accumulated in perinuclear organelles, and channel clustering practically disappeared from the surface membrane. This contrasts with rat myometrium, where there is a reduction of channel transcripts and overall protein levels including the surface membrane. We conclude that large conductance Ca2+-activated K+ channel surface expression is reduced in both rat and mouse late pregnant myometrium. However, in rats, the main mechanism for the reduced channel expression at the cell surface is a diminished transcription, whereas in mice, it is an altered traffic to the surface.