In situ hybridization finds many applications in modern pathology. In many cases, special attention is paid to the processing of the tissues prior to in situ hybridization. In order to investigate the value of RNA in situ hybridization (RISH) in retrospective studies, we performed RISH for calcitonin and calcitonin gene-related peptide (CGRP)-I and -II mRNA in eight medullary thyroid carcinomas processed in 1981-1983. RISH was successful with radioactive calcitonin and CGRP-I probes. With biotinylated probes, only calcitonin-specific probes gave adequate results. The concentrations of CGRP mRNA were probably too low to be detected by non-radioactive RISH. The results of RISH were correlated with the immunohistochemical localization of the polypeptides. The results matched in all cases except one, where hybridization for calcitonin mRNA was found, but no immunoreactive calcitonin polypeptide. We conclude that RISH can be successfully used for retrospective analysis, even after long storage of tissue embedded in paraffin.