In order to develop a comprehensive approach for the study of oxytocin (OT) and vasopressin (VP) gene expression in the rat hypothalamus, we first developed an intronic riboprobe to measure OT heteronuclear RNA (hnRNA) levels by in situ hybridization histochemistry (ISHH). Using this 84bp riboprobe, directed against intron 2 of the OT gene, we demonstrate strong and specific signals in neurons confined to the supraoptic (SON) and paraventricular (PVN) nuclei of the rat hypothalamus. We used this new intronic OT probe, together with other well established intronic and exonic OT and VP probes to reevaluate OT and VP gene expression in the hypothalamus under two classical physiological conditions, acute osmotic stimulation and lactation. We found that magnocellular neurons in 7-8 day lactating female rats exhibit increased OT but not VP hnRNA. Since VP mRNA is increased during lactation, this suggests that decreased VP mRNA degradation during lactation may be responsible for this change. In contrast, while there was the expected large increase in VP hnRNA after acute salt loading, there was no change in OT hnRNA, suggesting that acute hyperosmotic stimuli produce increased VP but not OT gene transcription. Hence, the use of both exon- and intronspecific probes which distinguish the changes in hnRNA and mRNA levels, respectively, can provide insight into the relative roles of transcription and mRNA degradation processes in changes in gene expression evoked by physiological stimuli.