Chronic exposure to gonadotropin-releasing hormone (GnRH) in vivo eventually results in a failure to maintain maximal gonadotropin secretion, suggesting a loss of pituitary responsiveness. To examine changes in pituitary sensitivity in vitro, we have developed a superfusion technique in which dissociated rat anterior pituitary cells are attached to Cytodex beads. In this system, continuous administration of 30 nM GnRH caused an initial 20-fold increase in LH and FSH secretion rates. However, hormone secretion declined rapidly, reaching basal levels within 12 h. Both the calcium ionophore A23187 and the cocarcinogen phorbol myristate acetate were effective in releasing additional LH and FSH from cells made refractory to 30 nM GnRH, whereas higher doses of GnRH and 8-bromo-cAMP were minimally active. A rapid loss of response was also seen with a superactive GnRH agonist but was not observed with a GnRH antagonist. Desensitization to prolonged superfusion with phorbol myristate acetate was also observed, although these cells remained sensitive to GnRH. In contrast to the development of tolerance produced by continuous exposure to GnRH, repeated delivery of GnRH in a pulsatile fashion maintained pituitary responsiveness for more than 24 h. A priming effect was observed for both LH and FSH. Such results demonstrate that continuous, but not intermittent, exposure to GnRH is capable of producing desensitization in superfused anterior pituitary cells.