In situ hybridization histochemistry with a probe directed against an intron sequence of the rat arginine vasopressin (AVP) gene was used to demonstrate localization and regulation of AVP heteronuclear RNA in discrete brain regions. Hybridization with an AVP intron I (AVPinI) probe revealed specific hybridization confined to cell nuclei of paraventricular nucleus, supraoptic nucleus (SON), and suprachiasmatic nucleus neurons of the rat hypothalamus. Grain counts revealed that the signal generated by the AVPinI probe represented 1.9% of that derived from an AVP exon C probe (AVPexC) in the SON. Interestingly, in the suprachiasmatic nucleus the proportion of AVPinI to AVP exon C ratio was much higher (12%), suggesting either increased transcription of the AVP gene or changes in posttranscriptional RNA processing. Regulatory experiments revealed that 2.6-fold increases in AVPinI signal could be visualized in the SON as little as 30 min after an acute salt load, a period during which no significant change in cytoplasmic AVP mRNA could be observed. In response to chronic salt loading, both AVP heteronuclear RNA and AVP mRNA were up-regulated. These data compared favorably with transcription rate values determined by nuclear run-on assay, suggesting that intronic in situ hybridization affords a relatively reliable method for assessment of rapid changes in gene transcription in individual central nervous system neurons.