The properties of gonadotropin-releasing hormone (GnRH) receptors were analyzed in isolated pituitary cells prepared by enzymatic dispersion with trypsin or collagenasehyaluronidase. The initial impairment of LH responses to GnRH in isolated cells prepared by both methods was reversed during culture for 2 days in multiwell vessels. However, specific binding sites for GnRH, assayed by equilibration with [125I]iodo-[D-Ser(t-Bu)6]des-Gly10 -GnRH N-ethylamide (GnRH-A) were demonstrable in collagenase-dispersed cells, both initially and after 2 days in culture. Cellular uptake of GnRH-A was temperature dependent, with rapid and saturable binding to a limited number of specific receptor sites with high affinity for the labeled analog (Ka = 4.0 ± 0.8 × 109 M-1). These sites showed common binding specificity for GnRH-A and the native GnRH peptide, with significantly lower affinity for the natural peptide (Ka = 2.3 × 107 M-1). Other protein and peptide hormones, including ovine LH, ovine PRL, hCG, TRH, somatostatin, and angiotensin II (up to 10-6M) did not inhibit binding of GnRH-A to its receptors. Cellular binding of GnRH-A was followed by increased cGMP production and LH release within 10 min. The analog was 50 times more potent than native GnRH in stimulating LH release. The Kact values derived for GnRH and GnRH-A were 0.5 and 0.01 nM, respectively, considerably lower than the Kd values of 50 and 0.25 nM derived from receptor binding analysis. These results indicate that GnRH receptors can be identified in isolated pituitary cells, in which peptide binding is followed by increased cGMP production and LH release. The impaired LH responses in acutely dispersed pituitary cells are not due to the loss of receptor sites but to a reversible postreceptor defect. Occupancy of about 20% of the GnRH-binding sites elicits a near-maximal LH response, indicating the nonlinearity of GnRH-receptor coupling to secretory responses in the cultured gonadotroph.