The neuronal growth-associated protein (GAP)-43 has been localized in both long fibers and punctate clusters by immunocytochemistry within the rat anterior pituitary (AP). After adrenalectomy (ADX), GAP-43 immunoreactivity (GAP-43-ir) is greatly increased and is associated with corticotrophs at the light microscopic level. We have undertaken an electron microscopic study to determine the cellular localization of GAP-43 in the post-ADX AP. Using preembedding immunocytochemistry, we found GAP-43-ir localized exclusively to the cytoplasmic surface of the plasmalemma within a subset of endocrine cells with ultrastructure typical of degranulated corticotrophs at 4 d after ADX. We combined preembedding immunoelectron microscopy for GAP-43 with immunogold labeling for ACTH and found that GAP-43-ir was invariably present only in cells containing ACTH-positive granules. The density of GAP-43-ir was highest within extensive processes emanating from the soma, suggesting that these processes are the basis for the punctate clusters of GAP-43 staining seen surrounding corticotrophs in the light microscope. We also observed rare synaptic-like contacts between GAP-43-ir processes and distant cell bodies. GAP-43 mRNA was detected in extracts of the AP 4 d after ADX using RT-PCR, and quantitative PCR confirmed that GAP-43 mRNA was significantly up-regulated in the AP in response to ADX. We postulate that increased expression of GAP-43 may stimulate process outgrowth and intercellular communication by activated corticotrophs.