Thyrotropin-releasing hormone (TRH) stimulates the prolactin (PRL) release from normal lactotrophs or tumoral cell line GH3. This effect is not observed in many patients with PRL-secreting tumors. We examined in vitro the PRL response to TRH on cultured human PRL-secreting tumor cells (n = 10) maintained on an extracellular matrix in a minimum medium (DME + insulin, transferrin, selenium). Addition of 10<sup>-8</sup> M TRH to 4 × 10<sup>4</sup> cells produced either no stimulation of PRL release (n = 6) or a mild PRL rise of 32 ± (SE) 11 % (n = 4) when measured 1 2 and 24 h after TRH addition. When tumor cells were preincubated for 24 h with 5 × 10<sup>-11</sup> M bromocriptine, a 47 ± 4% inhibition of PRL release was obtained. When TRH (10<sup>-8</sup> M) was added, 24 h after bromocriptine, it produced a 85 ± 25% increase of PRL release (n = 8). This stimulation of PRL release was evident when measured 1 h after TRH addition and persisted for 48 h. The half maximal stimulatory effect of TRH was 2 × 10<sup>-10</sup> M and the maximal effect was achieved at 10<sup>-9</sup> M TRH. When tumor cells were pretreated with various concentrations of triiodothyronine (T3), the PRL release was inhibited by 50% with 5 × 10<sup>-11</sup> M T3 and by 80% with 10<sup>-9</sup> MT3. Successive addition of TRH (10<sup>-8</sup> M) was unable to stimulate PRL release at any concentration of T3. The addition of 10<sup>-8</sup> M estradiol for up to 16 days either stimulated or had no effect upon the PRL basal release according to the cases. In all cases tested (n = 4), preincubation of the tumor cells with estradiol (10<sup>-8</sup> M) modified the inhibition of PRL release induced by bromocriptine with a half-inhibitory concentration displaced from 3 × 10<sup>-11</sup> M (control) to 3 × 10<sup>-10</sup> M (estradiol). These data demonstrate that the absence of TRH effect observed in some human prolactinomas is not linked to the absence of TRH receptor in such tumor cells. TRH responsiveness is always restored in the presence of dopamine (DA) at appropriate concentration. This TRH/DA interaction seems specific while not observed under T3 inhibition of PRL. Furthermore, estrogens, while presenting a variable stimulatory effect upon basal PRL, antagonize the dopaminergic inhibition of PRL release.