Studies were designed to assess the extent to which binding of follitropin (FSH) to its receptor is a reversible process. [125I]hFSH was allowed to interact with membrane receptors from calf testis for 2 h at various temperatures by which time significant specific binding of [125I]hFSH had taken place in all instances. Unlabeled FSH was then added (delayed addition) and the amount of [125I]hFSH remaining bound was monitored as a function of time. In order to assess reversibility, [125I]hFSH binding in the latter samples was compared to that occurring when unlabeled FSH had not been added or when unlabeled FSH was present from the start of the incubation. Binding was essentially fully reversible at 4 degrees C, but reversibility decreased with increasing temperature. Reversibility of FSH binding decreased markedly at temperatures greater than 26 degrees C and was considered irreversible at temperatures above 30 degrees C. At 4 degrees C essentially full reversibility (greater than 90%) was observed when the unlabeled hormone was added after 7 h of incubation, but decreased when added after 12 h. At warmer temperatures (22 or 30 degrees C) 'there was a progressive decrease in reversibility as the time of delay before addition of unlabeled hormone was lengthened. By determining the affinity constant (in separate experiments) at various temperatures, a thermodynamic analysis was possible. This analysis was restricted to the temperature range 4-26 degrees C in order to minimize complications arising from irreversible binding. The reaction was endothermal at low temperatures (T less than 12.5 degrees C) and exothermal at higher temperatures (T greater than 12.5 degrees C) and was associated with a decrease in heat capacity of 1800 cal [mol deg]-1 at 25 degrees C. The results are consistent with the concept that the hydrophobic effect plays an important role in FSH binding, but that the reaction is complex and may be composed of more than one step.