Isolated rat hepatocytes accumulate a slowly dissociable human growth hormone (hGH) binding fraction with incubation time. Slowly dissociable [125I]hGH is receptor bound, intact and immunocompetent. Fifty-six percent of the bound hormone was slowly dissociable within 3 min of the initiation of hGH-hepatocyte incubation. Subsequently, the proportion of slowly dissociable [125I]hGH increased at the expense of the rapidly dissociable fraction. This suggested that binding induced interconversion between different states of affinity of the hGH receptor. Preincubation with hGH diminished the capacity of hepatocytes to subsequently bind [125I]hGH. Receptor occupancy resulting from accumulation of slowly dissociable hGH accounted for 37 and 62% of the decreased binding after preincubation with 0.79 and 7.9 nM hGH, respectively. Fractional receptor occupancy, among but distinguishable from other processes, may account for the inverse relationship between site number and applied hormone concentration. Addition of hGH to the medium of [125I]-hGH-hepatocyte incubates increased the extent of loss of label from hepatocytes. The progressive retention of intact [125I]-hGH by hepatocytes with site occupancy and invariant receptor affinity subsequent to fractional saturation was inconsistent with negative cooperativity. A mechanism in which hGH diminished reassociation of [125I]hGH with available sites during dissociation was consistent with the available binding data. The interrelationship between peptide hormone in rapid and slow equilibrium with the medium is of fundamental importance in modulating receptor binding and availability.