Normal pregnancy is characterized by a dramatic increase in uterine blood flow, in part, due to augmented uterine vasodilatation. EDHF plays an important role in endothelium-mediated dilatation of uterine arteries; however, the nature of EDHF and its regulation by pregnancy remains largely unknown. The purpose of this study was to: (1) explore the role of small (SK) and intermediate (IK) conductance K⁺ channels in EDHF-dependent vasodilatation; (2) study vasodilator effects of K⁺ ions in uterine arteries of non-pregnant (NP) and late pregnant (LP) rats; (3) evaluate the role of K⁺ ions as EDHF. Rats were anesthetized with intraperitoneal injection of Nembutal and killed by decapitation. Segments of uterine radial arteries were cannulated and pressurized to 50 mmHg. Smooth muscle cells (SMCs) were loaded with fura 2 (5 µM); fura 2 fluorescence was measured using a photo-multiplier system. ACh- or K⁺-induced changes in arterial diameter and SMC Ca²⁺ ([Ca²⁺]_i) were studied in arteries pre-constricted with phenylephrine (PE) in the presence of 200 µM L-NNA and 10 µM indomethacin. ACh (0.03-10 µM) induced a dose-dependent dilatation that was greater in arteries from LP rats compared to NP rats (26 ± 6, n = 22 vs. 5 ± 2%, n = 21 at 0.03 µM ACh, P<0.01, ANOVA) and was associated with a more marked reduction in [Ca²⁺]_i (42 ± 12%, n = 3 vs. 11 ± 4%, n = 6, P<0.01). Combined treatment of NP or LP arteries with 100 nM apamin and 30 nM charybdotoxin (or 10 µM TRAM 34) abolished the ACh-induced reduction in [Ca²⁺]_i and vasodilatation. 5 mM K⁺ reduced [Ca²⁺]_i and dilated PE-pre-constricted NP arteries by 97 ± 2% (n = 5) and 92 ± 5% (n = 5) and LP arteries by 58 ± 10% (n = 5) and 60 ± 9%, (n = 8), respectively. K⁺-evoked changes in [Ca²⁺]_i and diameters were significantly smaller in vessels from LP rats compared to NP controls (P<0.05, unpaired t-test). K⁺-induced responses were incompletely inhibited with 50-100 µM BaCl₂ but were abolished by a combination of BaCl₂ and 10 µM ouabain. However, ACh-induced [Ca²⁺]_i reduction and vasodilatation were not affected by BaCl₂ and ouabain. These findings demonstrate that pregnancy augments EDHF-dependent dilatation of uterine arteries in response to ACh. Endothelial SK and IK channels are critically involved in EDHF-mediated uterine vasodilation. K⁺-induced vasodilatation is due to activation of both inward rectifier K⁺ channels and Na⁺-K⁺ pump. Although K⁺ ions can effectively dilate uterine arteries, they do not contribute significantly to the EDHF-mediated vasodilatation induced by ACh. These data suggested that electrotonic spreading of hyperpolarization from ECs to SMCs may be the underlying mechanism of EDHF in the maternal uterine circulation.