Fifteen cows (87 +/- 8 d in lactation; 641 +/- 33 kg BW) were randomly assigned to treatment and then subjected for 182 d to daily sc injection (1000 hr), in the cervical area, of saline (control), thyrotropin-releasing factor (TRF: 1 micrograms/kg BW), growth hormone-releasing factor (1-29)NH2 (GRF; 10 micrograms/kg BW) or GRF plus TRF (10 and 1 micrograms/kg BW, respectively) according to a 2 x 2 factorial design. On days 1, 31, 88 and 179, jugular blood samples were collected from 2 hr before to 6 hr after injection. Samples were also collected for 5 consecutive days after cessation of treatment. GRF always induced growth hormone (GH) release (600 vs 7925 ng.min/ml) with augmentation of response with time (interaction GRF * day; P less than .001). TRF did not affect (P greater than .25) GH release; there was no interaction (P greater than .25) with time. There was no significant interaction (P greater than .25) between GRF and TRF on GH release. However, the amount of GH release with GRF plus TRF was always greater than with GRF alone (9419 vs 6431 ng.min/ml). TRF induced a significant release of prolactin (23769 vs 42175 ng.min/ml) but GRF reduced the amount of prolactin release on the last day of sampling. TRF induced thyroid stimulating hormone (TSH) release only on the first day of injection while triiodothyronine (T3) and thyroxine (T4) continued to respond to TRF throughout the treatment period. Concentrations of T3 and T4 fell below control levels after cessation of TRF injection. In conclusion, GRF-induced GH release and TRF-induced Prl and thyroid hormone release were maintained over a 6-mo treatment period. TRF induced TSH release only on the first day of injection. Overall, these results raised the possibility of a direct effect of TRF on the thyroid gland.