Preincubation of rat pituitary cells in primary culture with rat GH-releasing factor (rGRF) resulted in substantial desensitization to subsequent GRF stimulation. rGRF-directed GH release and intracellular cAMP accumulation decreased in the desensitized cells. Whereas prior treatment of rat pituitary cells caused partial depletion of intracellular GH levels, diminished cellular reserves could not entirely account for the decreased GH release. Cells that had been preexposed to 10 nM rGRF for 4 h demonstrated at 30-50% depletion of intracellular GH; subsequent stimulation of those cells with 10 nM rGRF elicited GH release which was only 5% of that seen in cells that were not desensitized [control, 112 +/- 3.2 ng/well (+/- SEM); GRF-stimulated, 435 +/- 32 ng/well; GRF-pretreated, control, 63 +/- 3 ng/well, GRF-pretreated, GRF-stimulated, 73 +/- 3.4 ng/well]. Despite the marked depletion of cellular GH stores and the greatly diminished rGRF-stimulated GH release in cells that had been preexposed to rGRF, both forskolin and (Bu)2cAMP were able to induce a 2-fold stimulation of GH release. Incubation of the rGRF-pretreated cells with fresh medium which lacked rGRF resulted in gradual recovery of the ability of rGRF to stimulate GH release without complete reconstitution of the intracellular GH stores. These results indicate that exposure of rat pituitary cells to rGRF results in 1) partial depletion of intracellular GH stores; 2) a diminished ability of a subsequent rGRF challenge to elicit GH secretion and intracellular cAMP accumulation, and 3) a sustained ability of forskolin and (Bu)2cAMP to stimulate GH release, indicating that rGRF desensitization occurs in vitro.