The ability of myometrial β-adrenergic receptors to form a “high-affinity state” with β-adrenergic receptor agonists might be greater in pregnant guinea pigs at ≥0.9 of gestation than in nonpregnant animals. To determine whether this difference is due to pregnancy in general or is associated only with late pregnancy and to determine whether it persists in the postpartum period, we studied the interaction of I-isoproterenol with β-adrenergic receptors in myometrial membranes obtained from nonpregnant (nulligravid) animals, pregnant (primigravid) animals at 0.3, 0.7, and 0.9 to 1.0 of gestation (term 65 days), and postpartum guinea pigs (2 to 3 days). The affinity of myometrial β-adrenergic receptors for l-isoproterenol was measured by percent inhibition of −[ 125I]cyanopindolol binding. In the presence of magnesium chloride, the competition curves could be resolved into two affinity states of the P-adrenergic receptor, “high” and “low,” respectively, in all groups. The ratio of the dissociation constant of the “low”-affinity state to that of the “high”-affinity state was significantly higher in pregnant guinea pigs at ≥0.9 of gestation than in nonpregnant or postpartum animals and in pregnant animals of earlier gestations. In the presence of guanosine triphosphate only one (low-affinity) state of the receptor was detectable. Thus it is only in pregnant guinea pigs at ≥0.9 of gestation that the ability of myometrial β-adrenergic receptors to form a high-affinity state is enhanced.