Elevated neuropeptide Y (NPY) levels in the hypothalamus have been reported during lactation in the rat. The increase in NPY neuronal activity may be important in modulating a number of changes in hypothalamic neuronal function that are associated with lactation. The aims of the present study were to determine 1) if NPY neurons in the hypothalamus can be activated by the suckling stimulus; and 2) the time course of the activation in response to the suckling stimulus. In the first experiment, lactating rats were deprived of their 8-pup litters on day 9 post partum for 48 h. On day 11, the animals were divided into three groups and exposed to the suckling stimulus for varying periods of time up to 24 h. NPY neuronal activity was assessed by measuring changes in NPY messenger RNA (mRNA) levels, using in situ hybridization. NPY mRNA levels in the caudal portion of the hypothalamic arcuate nucleus (ARH) were approximately doubled by 24 h of suckling. NPY mRNA levels in the rostral portion of the ARH were not affected by suckling throughout the time examined. In addition to increased NPY mRNA in the ARH, resuckling for as little as 3 h induced NPY mRNA expression in cells located dorsal and lateral to the compact zone of the dorsomedial nucleus of the hypothalamus (DMH). NPY expression in these cells was not observed in the nonresuckled controls. These data demonstrate that the acute suckling stimulus activates two specific populations of NPY neurons in the hypothalamus: in the caudal portion of the ARH and in the DMH. The increased NPY neuronal activity may play an important role in modulating changes in hypothalamic regulation of hormone secretion and food intake.