The energy-dependent cyclic nucleotide cellular efflux is operative in numerous eukaryotic cells and could be mediated by multidrug resistance proteins MRP4, MRP5, and MRP8. In pituitary cells, however, the operation of export pumps and their contribution to the control of intracellular cyclic nucleotide levels were not studied previously. Here we show that cellular efflux of cyclic nucleotides was detectable in normal and immortalized GH(3) pituitary cells under resting conditions and was enlarged after concurrent stimulation of cAMP and cGMP production with GHRH, corticotropin-releasing factor, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and forskolin. In resting and stimulated cells, the efflux pumps transported the majority of de novo-produced cGMP, limiting its intracellular accumulation in a concentration range of 1-2 microm. In contrast, only a small fraction of cAMP was released and there was a time- and concentration-dependent accumulation of this messenger in the cytosol, ranging from 1-100 microm. Stimulation and inhibition of cGMP production alone did not affect cAMP efflux, suggesting the operation of two different transport pathways in pituitary cells. The rates of cAMP and cGMP effluxes were comparable, and both pathways were blocked by probenecid and progesterone. Pituitary cells expressed mRNA transcripts for MRP4, MRP5, and MRP8, whereas GH(3) cells expressed only transcripts for MRP5. Down-regulation of MRP5 expression in GH(3) cells decreased cGMP release without affecting cAMP efflux. These results indicate that cyclic nucleotide cellular efflux plays a critical role in elimination of intracellular cGMP but not cAMP in pituitary cells and that such selectivity is achieved by expression of MRP5.