Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.