The precise factors involved in the transition of the relaxed pregnant uterus to the contractile state at the onset of parturition remain unclear, but it is accepted that cAMP-generating pathways contribute to uterine relaxation. We have previously reported an increased expression of the adenylyl cyclase (AC)-stimulating protein Gαs in human myometrium during gestation, with a corresponding increase in GTP-stimulated AC activity. However, little is known about the predominating AC isoforms expressed during pregnancy. This information is important, because although all AC isoforms are stimulated by Gαs, their regulation by other signalling molecules is very different. In the present study we have identified the isoforms of AC expressed in both pregnant and non-pregnant myometrium by mRNA analysis and immunoblotting. mRNA encoding for AC I, II, III, VIII and IX was present in non-pregnant and pregnant myometrium, and in cultured myometrial cells. Differing levels of AC protein could be detected in myometrial plasma membranes, with decreased levels of Group 1 (isoforms I, III and VIII) and Group 4 (IX) ACs allied with increased levels of Group 2 (II, IV and VII) and 3 (V and VI) ACs during pregnancy. These findings imply a role for Group 2-activating pathways, eg G-protein βγ-subunits and protein kinase C, in the maintenance of uterine quiescence, whilst suggesting a lesser involvement of calcium–calmodulin complex, an activator of Group 1 AC isoforms, in uterine relaxation during gestation. These data may provide an alternative pharmacological approach for the attenuation of preterm labour.