Background and purpose: Uterine spontaneous contraction and pacemaking are poorly understood. This study investigates the role of the mitochondrial Ca(2+) store in uterine activity. Experimental approach: We investigated the effects of mitochondrial and sarco-endoplasmic reticulum (SER) inhibitors on contraction, membrane potential (Vm) and cytosolic Ca(2+) concentration ([Ca(2+) ](c) ) in longitudinal smooth muscle of the mouse uterus. Key results: The mitochondrial agents rotenone, CCCP, CGP37157 and kaempferol decreased the force of contractions. The ATP synthase inhibitor oligomycin had no significant effect. The effects of these agents were compared to those of SER inhibitors cyclopiazonic acid (CPA), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. All agents, except CPA and oligomycin, decreased contractile force. CPA and CCCP transiently increased contraction frequency, which returned to control levels, whereas rotenone, CGP37157, kaempferol and 2-APB decreased frequency and caffeine had no significant effect. Application of the mitochondrial agents when CPA functionally inhibited stores did not change contraction frequency but, with the exception of kaempferol, decreased force. CCCP caused depolarization and maintained increase in [Ca(2+) ](c) or depolarization/transient hyperpolarization and transient increase in [Ca(2+) ](c) for oestrus and di-oestrus tissues respectively. Rotenone caused hyperpolarization and maintained increase in [Ca(2+) ](c) . CGP37157 and kaempferol caused hyperpolarization but no measurable change in [Ca(2+) ](c) . Application of a range of K(+) channel blockers indicated a role of Ca(2+) activated K(+) (K(Ca) ) channels in the CCCP- and CGP37157-induced actions. Conclusions and implications: Mitochondria have a modulatory role on uterine contractions, with mitochondrial inhibition reducing contraction amplitude and pacemaker frequency by changes in Vm, [Ca(2+) ](c) and/or Ca(2+) influx.