5-HT(1A) modulation within the midbrain periaqueductal gray (PAG) is closely associated with anxiety- or panic-like behavior. Several findings have demonstrated that the properties of buspirone (a 5-HT(1A) partial agonist) would function as either anxiolytic or panicolytic in both clinical and laboratory animal research. In this study, we have investigated the neuronal activity occurring within the different regions of the PAG induced by buspirone treatment. Twenty-eight albino Wistar rats (350-400 g) were injected with either acute or chronic saline/buspirone (each, n=7), respectively. Our results show that buspirone treatment reduced locomotor activity, body weight and fecal boli, particularly in the chronic buspirone group. Two-way ANOVA revealed a significant decrease of c-Fos-immunoreactive (ir) cells expression in all regions of the rostral PAG after both acute and chronic buspirone (acute buspirone (AB) and chronic buspirone (CB), respectively) treatment. However, no effects on c-Fos-ir were detected in the caudal lateral periaqueductal gray (lPAG) and ventrolateral periaqueductal gray (vlPAG) in both the AB and CB groups, and in the dorsolateral periaqueductal gray (dlPAG) of the CB group. Interestingly, c-Fos-ir cells in the dorsomedial periaqueductal gray (dmPAG) column were reduced consistently in both the rostral and caudal PAG in both AB and CB groups. Besides, in all regions the number of c-Fos-ir cells was higher in the AB than in the CB group with exception of the rostral lPAG. In conclusion, the main anxiolytic effect of buspirone was specifically localized in all regions of the rostral PAG and in the caudal dmPAG. However, the caudal dlPAG, lPAG and vlPAG were found to be ineffective to buspirone treatment, probably due to their distinctive function in mediating higher level of anxiety in defensive behavior. This indicates that the longitudinal anatomical structure of the PAG possesses a different level of receptor sensitivity of 5-HT(1A) in the pathophysiology of anxiety and panic disorder.