This study demonstrates the response of human pituitary cell monolayers to a wide variety of hormonal stimuli. Appropriate release of luteinizing hormone (LH) and prolactin (PRL) was used as a verification of cell function. Cells that had been in culture for 20 days, with no hormonal additions, were exposed to LH-releasing hormone (LH-RH) continuously for 14 days. This resulted in an immediate fivefold increase in secretion of LH followed by a depression in LH production over the remaining 10-day period. After an 8-day period without hormonal additions, the same cultures again demonstrated a threefold increase in response to retreatment with LH-RH. In two similar studies, cells that had been in culture for 28 and 31 days were treated with bromocriptine, pergolide, dopamine, or thyrotropin-releasing factor (TRF). TRF elicited an increase of PRL in the medium by nearly double the control values. The addition of dopamine, pergolide, or bromocriptine resulted in a depression of PRL during the treatment period. This study has shown that human pituitary cells maintained in long-term monolayer culture respond predictably to a wide range of hormonal stimuli.