The classical mode of luteinizing hormone (LH) secretory desensitization in the rat appears after 3-6 h of continuous in vitro administration of gonadotropin (GnRH). A second mode has been reported to occur very rapidly (< 2 min) after the onset of GnRH administration, and to reverse within 3 min after its withdrawal. Here, the existence of a third mode of desensitization is reported. occurring at 40-50 min after initiation of continuous GnRH administration. Rat pituitary cells were perifused with 10(-8) M GnRH for 6 h: 10 min samples were collected for LH measurements by radioimmunoassay. As expected, the pattern of LH release was biphasic: LH levels peaked in the first phase at 30 min, decreased at 40-50 min, increased in the second phase to maximal levels at 90-110 min, and then decreased in the classical desensitization mode to near-baseline values by 300-360 min. Static incubations of pituitary cells in Petri dishes in the presence of high (10(-8) M) or submaximal (10(-9) M) GnRH concentrations confirmed the decrease in LH secretion at 40-50 min. Measurement of LH by reverse hemolytic plaque assay (RHPA) confirmed the existence of this new mode of desensitization; since 93% of all gonadotropes had become secretory at 40-50 min, the possibility of two subpopulations of gonadotropes accounting for the two phases of LH secretion appears to be ruled-out. GnRH receptor binding studies demonstrated a approximately 50% decrease in cell-surface binding in association with the desensitization at 40-50 min. These studies suggest the existence of a third mode of GnRH-induced LH secretory desensitization that is not due to gonadotrope subpopulations but may be causally associated with decreased GnRH receptor binding.