The effect of inhibiting myosin light chain kinase on contractions of human and rat myometrium has been investigated, to determine whether force can be produced independently of myosin phosphorylation. Two inhibitors were used, wortmannin and ML-9, and their effects on spontaneous, high-K-depolarization-induced and oxytocin-induced force studied. Both inhibitors reduced and then abolished uterine force, irrespective of how it was produced; this was the case for both human and rat myometrium, and pregnant and non-pregnant tissue. The effects of wortmannin on intracellular [Ca2+] and inward Ca2+ current were examined. The data showed that the reduction in force produced by wortmannin occurs without a reduction of either the Ca2+ current or [Ca2+]. It is concluded that, under normal physiological conditions, myosin light chain kinase phosphorylation of myosin is essential for uterine force production and that there is little or no role for alternative force-producing pathways.