Spontaneous bladder contractions (SBCs) in the neonatal rat urinary bladder change from a high-amplitude, low-frequency pattern to a low-amplitude, high-frequency pattern during the first 6 weeks of life. Understanding the mechanism of this developmental change may provide insights into causes of bladder overactivity in adults. In vitro whole bladder preparations from Sprague-Dawley rats were used to study the modulation of SBCs by calcium-activated potassium channels (K_Ca) and electrical field stimulation from 3 days to 6 weeks of life. SBCs in 3 day bladders were unmasked by treatment with iberiotoxin (100 nM), an inhibitor of large conductance K_Ca (BK) channels, or apamin (100 nM), an inhibitor of small conductance K_Ca (SK) channels. Iberiotoxin significantly increased the magnitude of SBCs at 2-3 weeks, whereas apamin was only effective at 6 weeks. In 1-2 week bladders, exposure to room temperature Krebs solution decreased SBCs. This decrease was reversed by activating intramural nerves with electrical field stimulation. The effect of electrical field stimulation was inhibited by atropine (1 µM), suramin (10 µM) or pretreatment with tetrodotoxin (1 µM), but was not reversed by tetrodotoxin applied after electrical field stimulation. BK mRNA increased 3 fold, and BK protein increased 5 fold from 3 days to 6 weeks. These data suggest that BK channels play an important role in the regulation of SBCs in the neonatal bladder, and that both increased BK channel activity as well as changes in smooth muscle sensitivity to locally released neurotransmitters contribute to the downregulation of SBCs during early postnatal development.