Human and rat placentas contain enzymatic activity which converts T₄ to rT₃ and T₃ to 3,3′-diiodothyronine and 3′-monoiodothyronine. This study presents data on the ontogeny of this inner ring iodothyronine deiodinase activity (P-T₄ase) in rat placenta. P-T₄ase was measured by quantitating the conversion of T₄ to rT₃ in 700 × g supernatants of placental homogenates. Groups of rats were mated to permit the dams to be killed on the same day, on days 12, 14, 16, 18, and 20 of gestation. Sufficient placental tissue was obtained to measure P-T₄ase on all but the 12th day of gestation. The highest level of P-T4ase was observed on day 16. P-T₄ase on days 14, 18, and 20 was 52%, 77%, and 41%, respectively, of that observed on day 16 (P < 0.01, day 16 vs. all other days). Amniotic fluid rT₃ concentrations were highest on day 18 and were 61% and 64%, respectively, of that observed on day 18 (P < 0.01, days 16 and 20 vs. day 18). At 20 days, maternal serum T₄ concentrations were significantly lower (P <0.01) than on days 14, 16, or 18. A brief period of maternal hypothyroidism (4 or 9 days before the time that the animals were killed on day 20 of gestation) did not significantly alter P-T₄ase. These studies indicate that there are age-dependent changes in placental inner ring deiodinase activity in the rat. Amniotic fluid rT₃ concentrations may reflect these changes. Brief reductions in maternal serum T₄ concentrations do not account for changes in placental inner ring deiodinase activity. These studies emphasize the importance of gestional age in studies of placental inner ring iodothyronine deiodinase.