Several protein components that are essential for exocytotic membrane fusion in neurons have recently been identified. The expression and cellular localization of such protein components were examined in the rat pituitary gland. In situ hybridization using isoform-specific oligonucleotide probes to different exocytotic protein messenger RNAs (mRNAs) showed strong hybridization signal for synaptotagmin I, cysteine string protein (CSP), VAMP-2 (vesicle-associated membrane protein), cellubrevin, munc-18 (mammalian homologue of unc-18), SNAP-25a (synaptosomal-associated protein of 25 kDa), syntaxin 1A, syntaxin 4, syntaxin 5, and alpha-SNAP (soluble NSF attachment protein) in the anterior and intermediate, but not in the posterior lobe of the pituitary. Moderate to weak hybridization signal was detected for synaptotagmin III, SNAP-25b, and syntaxin 2 mRNA in the anterior and intermediate, but not in the posterior lobe of the pituitary. Synaptotagmin II, VAMP-1, syntaxin 1B, or syntaxin 3 mRNA expression could not be detected in any part of the pituitary gland. Immunofluorescence histochemistry in combination with confocal laser microscopy revealed that synaptotagmin-, VAMP-, CSP-, NSF-, and alpha-SNAP-like immunoreactivities (-LI) were present in granules of cells in the anterior and intermediate lobe, whereas SNAP-25- and syntaxin-LI were primarily located to the plasma membrane. Synaptotagmin-, VAMP-, CSP-, NSF-, alpha-SNAP-, SNAP-25- and syntaxin-LI were all present in nerve fibers of the posterior lobe. Within cells of the anterior lobe, colocalization could be demonstrated for synaptotagmin I/II- and synaptotagmin III-LI with ACTH-, GH-, PRL- and TSH-, but not FSH- or LH-LI, whereas VAMP, CSP-, NSF-, alpha-SNAP-, SNAP-25- and syntaxin-LI were demonstrated in all hormone-containing cell types of the anterior pituitary. The results show the presence of several protein components and their isoform-specific mRNAs in the rat pituitary gland, suggesting that these proteins, similar to their roles in regulation of synaptic neurotransmitter release, may participate in exocytotic events in endocrine pituitary cells and in neurosecretory nerve endings of the neurohypophysis.