We investigated whether β-adrenergic receptors (β-AR) regulate the phospholipase C (PLC) system in midpregnant rat myometrium. PLCβ isoforms were characterized, and the effect of isoproterenol (β-adrenergic agonist) was tested on myometrial inositol phosphate (InsP) production and uterine contraction. Using specific antibodies, we showed that rat myometrium expresses PLCβ1, PLCβ3, and PLCβ4, and to a lesser degree PLCβ2. Quantitative analysis revealed that PLCβ isoforms are differentially expressed during pregnancy. Indeed, the amount of PLCβ4 is increased at midpregnancy, whereas PLCβ1, PLCβ2, and PLCβ3 are up-regulated at term. At midpregnancy, pretreatment of myometrial strips with isoproterenol significantly reduced basal and agonist-stimulated InsP production. Forskolin, a diterpene that increases cAMP accumulation by directly activating adenylyl cyclases, had no effect on InsP production. In contrast, two global potassium (K ) channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), prevented attenuation of InsP production by isoproterenol. Isoproterenol also significantly decreased spontaneous and agonist-induced contraction of the longitudinal layer of midpregnant rat myometrium. Preincubation of uterine strips with TEA plus 4-AP prior to β-AR activation blocked only partial uterine relaxation, whereas Forskolin was as potent as isoproterenol. This indicates that β-AR operate through both K channels and cAMP to induce uterine relaxation. In conclusion, we show for the first time that three myometrial PLCβ isoforms (PLCβ1, PLCβ2, and PLCβ3) are down-regulated at midpregnancy. At this period, β-AR reduce basal and agonist-stimulated InsP production through activation of K channels. Altogether, these mechanisms could act to decrease responsiveness of the longitudinal layer of myometrium to contractant factors.