Injections of adult Fundulus heteroclitus with 0.2 IU of oTSH over 4 days caused an increase in serum T4, with no effect on serum T3. Administration of 12 IU of oPRL in conjunction with the same dosage of TSH prevented the TSH-induced rise in T4, without affecting serum T3 levels. The failure of TSH to cause T4 levels to rise in the presence of PRL may result from either inhibition of release or acceleration of metabolic clearance of T4. We therefore conducted three experiments to examine potential effects of PRL on the kinetics of peripheral clearance of thyroid hormones. 125I T4 was cleared from serum in a biphasic pattern that was unaltered by PRL. Clearance of labeled T3 followed a similar pattern that was also not influenced by PRL treatment. Generation of labeled T3 by deiodination of a dose of 125I T4 was quantified over a 24-h period. Again, PRL-treated fish showed no significant differences. Since PRL is without effects on thyroid hormone clearance patterns or deiodination rate, we conclude that PRL prevents TSH-induced increases in serum T4 in this species by directly affecting thyroid function.