The physiological roles of TRH in pituitary lactotrophs, particularly during lactation, remain unclear. We studied the prolactin (PRL) status, including serum PRL and PRL mRNA levels in the pituitary, in nonlactating and lactating TRH-deficient (TRH−/−) mice with a rescue study with thyroid hormone and TRH. We found that, as reported previously for male TRH−/− mice, neither the morphology of the lactotrophs, PRL content in the pituitary, nor the serum PRL concentration was changed in nonlactating female TRH−/− mice. However, concurrent hypothyroidism induced a mild decrease in the PRL mRNA level. In contrast, during lactation, the serum PRL level in TRH−/− mice was significantly reduced to about 60% of the level in wild-type mice, and this was reversed by prolonged TRH administration, but not by thyroid hormone replacement. The PRL content and PRL mRNA level in the mutant pituitary during lactation were significantly lower than those in wild-type mice, and these reductions were reversed completely by TRH administration, but only partially by thyroid hormone replacement. Despite the low PRL levels, TRH−/− dams were fertile, and the nourished pups exhibited normal growth. Furthermore, the morphology of the pituitary was normal, and high performance gel filtration chromatography analysis of the PRL molecule revealed no apparent changes. We concluded that 1) TRH is not essential for pregnancy and lactation, but is required for full function of the lactotrophs, particularly during lactation; and 2) the PRL mRNA level in the pituitary is regulated by TRH, both directly and indirectly via thyroid hormone.