Using a double-label immunofluorescence method, we analyzed the time course of the appearance of Fos and Jun in the nuclei of supraoptic nucleus (SON) neurons following intraperitoneal injection of hypertonic saline. Fos and Jun immunostaining appeared within 30 min, peaked at 90-120 min, and disappeared 4-5 h later. At all time points (30, 60, 120, 180, 240 min postinjection), colocalized Fos and Jun immunostaining was observed (> 90% colocalized staining in labeled neurons). At 4 h post-saline injection, some rats received a second injection of normal or hypertonic saline. A second injection of normal saline resulted in no Fos/Jun immunostaining 90 min later, while hypertonic saline induced combined Fos/Jun staining in only 17% of SON neurons. Of the remaining SON cells, 23% had staining to Fos alone and 4% of the cells stained for Jun only. In spite of the delivery of an effective second osmotic stimulus, determined by assessment of plasma osmolality and sodium content, SON neurons exhibited less colocalized Fos/Jun immunostaining, dramatically less Jun expression, and substantial, but attenuated, immunostaining for Fos. These results are discussed in the context of known negative feedback mechanisms that control the re-expression of these transcription factors.