The influence of TRH pulse pattern on PRL and TSH alpha- and beta-subunit gene expression was examined in vitro. Pituitaries from adult female rats were dissociated and plated for 48 h to allow attachment to collagen-coated microcarrier beads. The beads were perifused for 24 h with TRH (pulses or continuous). To examine the effects of TRH pulse amplitude, TRH pulses (0.032-100 nM) were given every 60 min (controls received either medium pulses or continuous 100 nM TRH). PRL mRNA rose progressively to a peak at 4 nM TRH/pulse (93% increase vs. medium-pulsed controls), but higher TRH pulse amplitudes were less effective. alpha-Subunit mRNA also rose with increasing TRH pulse dose, with maximal (137%) elevations after 100-nM pulses. TSH beta mRNA concentrations were increased by TRH pulse doses between 0.8-20 nM, but a clear dose-response pattern was not seen. Continuous TRH (100 nM) resulted in PRL, TSH beta, and alpha mRNAs that were less than the values in medium-pulsed controls. To assess the effects of pulse frequency, 4-nM TRH pulses were given at intervals between 15-240 min (controls received medium pulses or continuous 4 nM TRH). PRL mRNA was increased (55-107% increase) after all pulse intervals, except 240 min, and rose to a similar degree after 4 nM TRH given continuously. alpha-Subunit mRNA concentrations increased by a lesser degree and also did not rise after the slowest (240 min) pulse interval. In contrast, TSH beta mRNA levels increased progressively as pulse intervals were increased, and maximal (85%) elevations were seen after 240-min pulses. Continuous 4 nM TRH did not alter alpha or TSH beta mRNAs. These results show that pulsatile TRH is more effective than continuous TRH in stimulating PRL, TSH beta, and alpha mRNAs. Further, the pattern of TRH pulsatile signals can influence the expression of these pituitary hormone genes in a differential manner.