The prolactin immunoreactive neurons of the rat lateral hypothalamus were previously reported to express the dynorphin and secretogranin II genes. In the present study, the response of these neurons to osmotic challenge was immunocytologically investigated by using prolactin, dynorphin, secretogranin II and c-Fos antisera. In addition, the mRNA levels for secretogranin II and dynorphin were compared by in situ hybridization in controls and salt-loaded rats. For this model of chronic hyperosmolality, the prolactin and c-Fos immunoreactivities were not stimulated by salt drinking, but dynorphin and secretogranin II immunoreactivities as well as mRNA levels for dynorphin and secretogranin II significantly increased in the lateral hypothalamus. We suggest that the prolactin-immunoreactive neurons may be involved in the regulation of water homeostasis.