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BACKGROUND: Calretinin and parvalbumin are members of the intracellular calcium binding protein family, which transform Ca2+ bioinformation into regulation of neuronal and neural network activities. OBJECTIVE: To observe expression and co-expression of calretinin and parvalbumin in rat facial nucleus neurons. DESIGN, TIME AND SETTING: Neuronal morphology experiment was performed at the Research Laboratory of Applied Anatomy, Department Neurobiology and Anatomy, Xiangya Medical College of Central South University from August to October 2007. MATERIALS: Five healthy, adult Sprague Dawley rats were selected. Polyclonal rabbit-anti-parvalbumin and mouse-anti-calretinin were provided by Sigma, USA. METHODS: Rat brains were obtained and cut into coronal slices using a freezing microtome. Slices from the experimental group were immunofluorescent stained with polyclonal rabbit-anti-parvalbumin and mouse-anti-calretinin antibodies. The control group sections were stained with normal rabbit and mouse sera. MAIN OUTCOME MEASURES: lmmunofluorescent double-staining was used to detect calretinin and parvalbumin expression. Nissi staining was utilized for facial nucleus localization and neuronal morphology analysis. RESULTS: The majority of facial motor neurons was polygon-shaped, and expressed calretinin and parvalbumin. The calretinin-immunopositive neurons also exhibited parvalbumin immunoreactivity, that is, calretinin and parvalbumin were co-expressed in the same neuron. CONCLUSION: Calretinin and parvalbumin were expressed in facial nucleus neurons, with varied distribution.