This study examines the developmental expression of GABAB receptor subunits (GABAB(1a), GABAB(1b), GABAB(2)) in the pituitary intermediate lobe using in situ hybridization, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blots. Receptor functionality was studied by baclofen-stimulated GTPgammaS binding. In the adult rat pituitary all three transcripts were detected in melanotropes, but not in glia, of the intermediate lobe. No transcripts of any subunit were detected in the neural lobe. Transcripts of GABAB(1a) and GABAB(1b), but not of GABAB(2), were detected in specific subpopulations of cells in the anterior lobe. All three transcripts were detected in melanotropes on gestational day 18 using in situ hybridization. Reverse transcriptase-polymerase chain reactions comparing postnatal day 2 and adult transcript levels in the neurointermediate lobe support in situ hybridization data that GABAB(1a) mRNA levels do not change, GABAB(1b) levels increase, and GABAB(2) levels decrease as the rat matures. Thus, GABAB receptor subunit transcripts are differentially regulated in melanotropes during development. In the adult rat both GABAB(1) and GABAB(2) proteins were detected in the neurointermediate lobe using Western blotting and in melanotropes by immunohistochemistry. Developmentally, GABAB(1) protein was not detected until postnatal day 7, but was clearly expressed by postnatal day 15 while GABAB(2) protein could not be detected until postnatal day 15. Functional receptors were found in the intermediate lobe at postnatal day 15 and in the adult. The demonstration of transcripts for GABAB(1a), GABAB(1b) and GABAB(2) subunits at gestational day 18 contrasted with the failure to detect any protein before postnatal day 7, suggesting that the regulation of GABAB subunit isoforms occurs differentially at both the transcriptional and translational level as development progresses. The disparity in the regulation of the receptor subunits may suggest that GABAB(1) could have other functions besides being part of the GABAB receptor heterodimer.