We have used specific radioimmunoassays coupled with reversed-phase high-performance liquid chromatography (HPLC) to measure and characterise substance P (SP) and substance K (SK) in subdivisions of the rat hypothalamus. SP and SK levels in the paraventricular nucleus (PVN) were 968 +/- 61 and 381 +/- 22 pg respectively; in the supraoptic nucleus (SON) were 210 +/- 21 and 79 +/- 8 pg; and in the median eminence/arcuate nucleus (ME) were 1044 +/- 66 and 451 +/- 20 pg. Reversed-phase HPLC revealed that immunoreactive (ir) SP was present solely in the non-oxidised form in all tissue extracts. The principal form of ir-SK in the PVN and SON coeluted with synthetic SK on HPLC, but some immunoreactivity eluted in a later position. This material represented less then 5% of the total ir-SK in extracts of the PVN and SON, but increased to 35-40% of the total in the ME. Gel chromatography and HPLC characterised this compound as being slightly smaller and more hydrophobic than SK. These results establish that ir-SK is present within the hypothalamus in varying amounts and molecular forms. The location of significant amounts of both SP and SK in the PVN and ME, the principal regions of CRF-41 synthesis and release, is compatible with a role for neurokinins in the modulation of CRF-41 and consequently ACTH release.