The magnocellular oxytocin neurons within the paraventricular and supraoptic nuclei (PVN and SON) of the hypothalamus are important relays in the milk ejection reflex in lactating animals, and are activated by suckling. It has been suggested that proto-oncogene transcription factors such as Fos/Jun act as early nuclear transducers of sensory stimuli in neurons. Therefore, we have studied with immunohistochemistry Fos-related antigens (FRAs) as a marker for neuronal activity in the PVN and SON during suckling in lactating rats. In nonlactating rats, only few cells exhibiting FRAs were observed in these nuclei. Also in lactating rats subjected to continuous suckling Fos-like activity was low. In contrast, lactating rats separated from their pups for 4 h and then exposed to suckling for 1 h expressed strong Fos-like immunoreactivity, both in vasopressin and oxytocin neurons. Using in situ hybridization and immunohistochemistry we have also investigated the expression of the mRNAs for oxytocin, dynorphin, galanin and galanin message-associated peptide and of oxytocin and dynorphin in the PVN of lactating and nonlactating rats. In lactating rats, an increase in oxytocin and dynorphin and their mRNAs was observed, whereas mRNAs for galanin and galanin message-associated peptide were downregulated. With the help of immunohistochemistry and double-staining methods, a substantial coexistence between oxytocin- and dynorphin-like immunoreactivities was shown in magnocellular neurons. These results indicate that FRAs are activated in the PVN in the beginning of a suckling period, while this response cannot be seen after continuous stimulation. Furthermore, in the PVN of lactating rats, an upregulation of oxytocin and dynorphin occurs while galanin expression decreases. Finally, the coexistence between oxytocin and dynorphin is more pronounced in lactating rats and nonlactating female rats than has previously been described in male rats.