Caveolae are membrane organizing centres that recruit proteins and lipids for participation in intracellular trafficking and signal transduction. Our recent findings have revealed both a structural (Chanrachakul et al. 2003a,b) and functional (Chanrachakul et al. 2004) associations between the large conductance calcium-activated potassium (BK_Ca) channel and the β₂ adrenergic receptor (AR) in mediating uterine relaxation. The aim of this study was to investigate whether the BK_Ca channel/ β₂ AR complex and L type calcium (Ca²⁺) channels are localized together with caveolae of pregnant human myometrium in order to regulate uterine excitability. This study was approved by Derbyshire Research Ethics Committee and written informed consent was obtained from each participant. Myometrial biopsies were taken from term pregnant women (n = 5) undergoing elective Caesarean section. Myometrial cells were enzymatically dispersed and grown in culture media. Cultured human myometrial cells were then fixed and indirect double immunofluorescence staining was performed. Mouse monoclonal anti-caveolin-1 and rabbit polyclonal anti-BK_Ca channel, β₂ AR and L type Ca²⁺ channel were used in this study. Confocal microscopy was used to examine the association between the BK_Ca channel, β₂ AR and the L type Ca²⁺ channel with caveolin-1 in isolated pregnant human myometrial cells. Confocal immunofluoresecence demonstrated strong labelling of BK_Ca channels, β₂ AR, and L type Ca²⁺ channels in uterine myocytes. Caveolin-1 was located both on the membrane and intracellularly. Double staining immunofluorescence analysis indicated colocalization between BK_Ca channels, β₂ AR, and L type Ca²⁺ channels with caveolin-1 in primary cultures of human myometrial cells. These results suggest that the L type Ca²⁺ channels might be linked with the BK_Ca channels/ β₂ AR complex and are localized together in caveolar microdomains of pregnant human myometrial cells. Further studies are underway to examine the role of this compartmentation in regulating uterine activity.