Aim: The Tweety (Ttyh1-3) and Bestrophin (Best1-4) gene families produce calcium activated chloride currents (I_CaCl) when heterologously expressed. Whilst unlikely to solely encode calcium-activated chloride channels (CACCs), their contribution to smooth muscle I_CaCl is unknown as there is no information on whether any member of the Tweety or Bestrophin family is expressed in smooth muscle. Consequently, the present study aimed to ascertain the expression profile of these two gene families in murine smooth muscle with particular focus on those tissues where robust I_ClCa have been recorded. Methods: Whole-cell patch clamp electrophysiological recordings, semi-quantitative and quantitative reverse transcriptase polymerase chain reaction experiments and immunological studies were performed on various murine (BALB/c and C57; 6 – 8 weeks) smooth muscle tissues. Results: Cl- currents with distinctive voltage-dependent characteristics were recorded from myocytes isolated from murine portal vein, thoracic aorta, carotid artery and uterine cells (n>=5). Tweety and Bestrophin genes were detected in vascular and non-vascular smooth muscle samples (n=3). Interestingly, relative expression levels of the Best 1-3 mRNA were hard to detect in BALB/c tissues than in comparible tissues from C57 mice. Immunodetection of Bestrophin with two different Bestrophin antibodies revealed a number of bands suggesting this protein is susceptible to post-translational modification. Moreover, sucrose gradient experiments suggest Bestrophin may localise to lipid microdomains. Conclusion: This study is the first to demonstrate the presence of mRNA and protein and mRNA encoding the Best and Tweety gene family members, respectively, in a variety of vascular and non-vascular smooth muscle tissues. In addition we show an interesting strain difference in expression levels of Bestrophin family members.