The presence of type-II thyroxine 5'-deiodinase (5'-D) activity in rat pineal gland has been previously described. In the present paper, 5'-D activity, N-acetyltransferase (NAT) activity, and melatonin content were measured in the same rat pineal. Each of these constituents exhibits a nocturnal increase with peak values at 0100 h for melatonin (1.20 +/- 0.12 ng/gland) and at 0300 h for both 5'-D (39.5 +/- 11.9 fmol/gland/h) and NAT (8.38 +/- 1.04 nmol/gland/h) activities. In vivo treatment with iopanoic acid (IOP) completely prevented the nocturnal increase in 5'-D activity (14.1 +/- 2.6 fmol/gland/h at 0300 h) with no modification in either the NAT activity or melatonin content. Thyroidectomy greatly enhanced the 5'-D activity during the dark period (102.9 +/- 10.2 vs. 31.6 +/- 4.2 fmol/gland/h), reaching a peak at 0200 h; thyroidectomy, however, did not affect daytime pineal 5'-D activity (3.11 +/- 0.78 vs. 2.5 + 0.92 fmol/gland/h). Treatment of rats with IOP acid completely inhibited the pineal 5'-D activity in both control (7.86 +/- 0.88 fmol/gland/h) and thyroidectomized animals (2.24 +/- 1.10 fmol/gland/h) with no change in the melatonin content of the gland (1.21 +/- 0.32 vs. 0.99 +/- 0.18 ng/gland).