There is strong evidence that kisspeptin acts to regulate gonadotrophin-releasing hormone (GnRH) secretion, but whether there is also a component of action on the gonadotropes is not clear. Using quantitative RT-PCR, we found that GPR54 mRNA is expressed in ovine pituitary cell fractions enriched for gonadotropes as well as in somatotropes and lactotropes. To test whether kisspeptin acts directly on the pituitary gonadotropes, we first examined luteinising hormone (LH) release from primary ovine pituitary cell cultures treated with kisspeptin. We found that kisspeptin treatment increased the concentration of LH in culture media by 80% compared to control, but only in pituitary cultures from ewes during the follicular phase of the estrous cycle. Following this, we determined whether kisspeptin acts on the pituitary gland in vivo. Using GnRH-replaced ovariectomized hypothalamo-pituitary-disconnected (HPD) ewes, we were not able to achieve any effect of kisspeptin on LH under steady-state conditions or during the period of an estrogen-induced LH surge. Finally, we collected hypophysial portal blood samples from ovariectomized ewes and measured kisspeptin levels. Low but detectable amounts of kisspeptin were found in portal plasma, but levels were similar in ovariectomized ewes that were untreated or given estrogen to elicit an LH surge. Thus, although we observed an effect of kisspeptin on LH release in vitro in some situations, similar findings were not obtained in in vivo. Moreover, the low concentrations of kisspeptin in hypophysial portal blood and the lack of any change during the period of an estrogen-induced GnRH/LH surge, suggests that action on the pituitary gland is not of major consequence in terms of LH release.