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We have investigated the effect of the time of culture on cell number, cell content of LH, cell responsiveness to gonadotrophin releasing hormone (GnRH) and binding parameters of GnRH in rat anterior pituitary cells in culture. Although a decrease in the cell number was observed during the culture period, the cell content of LH remained unchanged. The receptor affinity (K_a) in acutely dispersed cells was 0·86 x 10⁷l/mol for [³H]GnRH and 1·36 x 10¹⁰l/mol for a highly potent agonist, D-Ser(Bu^t)⁶]GnRH(1–9)nonapeptide-ethylamide (GnRH-A). The affinity and binding capacity (0·3 fmol/10⁶ cells) for iodinated GnRH-A did not change significantly during the 6-day culture period. On the contrary, the values of K_a and binding capacity (257 fmol/10⁶ cells) for tritiated GnRH decreased by about 50% betweeen days 1 and 6 of culture. Our results suggest that ¹²⁵I-labelled GnRH-A binds mostly to high-affinity and low-capacity receptor sites, while [³H]GnRH, which must be used at a higher concentration, also binds to low-affinity, high-capacity binding sites and is therefore useless for the measurement of GnRH receptor binding affinity and binding capacity. Since the biological response of the cells to GnRH increased with the time of culture, it is concluded that although GnRH action is receptor-mediated, binding capacity and biological activity are not necessarily correlated.