A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. After PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE)-mediated degradation. We used ductus from immature (n = 25) and mature (n = 21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activity that contributes to the decreased sensitivity of the late-gestation ductus arteriosus to vasodilators like PGE2.