Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in the molecular regulation of neurotransmitter release. SNAP-25 exists in two isoforms, SNAP-25a and SNAP-25b, which arise from alternate splicing and which are differentially expressed throughout the nervous system. In situ hybridization was used to examine the presence and subcellular localization of SNAP-25a and SNAP-25b RNA transcript expression in motor and parasympathetic nuclei associated with cranial nerves. SNAP-25a RNA transcripts were strongly expressed in the parasympathetic Edinger-Westphal nucleus and dorsal motor nucleus of the vagus nerve but weakly expressed in motor nuclei such as the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal and accessory nuclei and in motoneurons of mouse lumbar spinal cord. In contrast, SNAP-25b RNA transcripts were not detectable in the Edinger-Westphal nucleus and dorsal motor nucleus of the vagus nerve but were strongly expressed in the oculomotor, trochlear, trigeminal, facial, ambiguus, hypoglossal, and accessory nuclei and in the motoneurons of mouse lumbar spinal cord. In the parasympathetic cranial nerve nuclei, displaying high levels of SNAP-25a RNA transcripts, the labeling was cytoplasmic, whereas the labeling was nuclear in the cranial nerve motor nuclei, displaying lower levels of transcripts. In contrast, labeling of SNAP-25b RNA transcripts was cytoplasmic in cranial nerve motor nuclei and not detectable in parasympathetic cranial nerve nuclei. Possible explanations for the region-specific and differential subcellular localization of SNAP-25a and SNAP-25b RNA transcripts are discussed.