In this study we investigated the neurochemical identity of the arcuate cells activated following GH-releasing peptide-6 (GHRP-6) injection by comparing, on consecutive sections, the distribution c-fos messenger RNA (mRNA) with that of mRNAs for peptides synthesized in arcuate cells, including neuropeptide Y (NPY), GH-releasing factor (GRF), tyrosine hydroxylase, POMC, and somatostatin. Rats bearing chronically implanted jugular catheters were injected with either 50 micrograms GHRP-6 or vehicle. Thirty minutes later they were terminally anesthetized and perfused with fixative. Paraffin-embedded sections of 7 microns thickness were processed using in situ hybridization for either c-fos mRNA or mRNAs for the neurochemical markers. In GHRP-6-treated rats the mean (+/-SEM) number of cells expressing c-fos mRNA in the arcuate nucleus (23 +/- 2 cells/section per rat; n = 5) was significantly higher than for vehicle-treated controls (2 +/- 1 cells/section per rat; n = 5; P < 0.001, Mann-Whitney U test). Superimposed camera lucida maps indicated that, in GHRP-6-injected rats, neurochemically identifiable cells expressing c-fos mRNA also express NPY mRNA (51 +/- 4%), GRF mRNA (23 +/- 1%) tyrosine hydroxylase mRNA (11 +/- 3%), POMC mRNA (11 +/- 2%), or somatostatin mRNA (4 +/- 1%). Thus, the majority of cells expressing c-fos mRNA following GHRP-6 injection are NPY and GRF-containing cells.