OBJECTIVE To study the cellular distribution of CD44 mRNA transcripts in tissue sections of colorectal cancer and corresponding normal colonic mucosa in order to correlate the findings with information from immunohistochemical methods and previous data from analysis by reverse transcription-polymerase chain reaction (RT-PCR). METHODS In situ hybridisation (ISH) analysis of CD44 standard (CD44s) and variant (CD44v) mRNA in cryostat sections of normal and neoplastic colonic mucosa with 35S-labelled riboprobes. Immunohistochemistry was performed on cryostat sections from the same patients using monoclonal antibodies directed against epitopes encoded by CD44 exon 1 (F.10.44.2), exon 5, (Hermes 3), exon 7 (23.6.1), and exon 11 (2F10). RESULTS CD44s and CD44v transcripts were both strikingly increased in carcinomas compared with corresponding normal mucosa and the abundant CD44v transcripts in tumour tissues were localised exclusively in the cancer cells. CD44s transcripts were present in cancer, inflammatory and resident stromal cells, but the relative amount in carcinoma cells was greater. Immunohistochemical staining broadly paralleled these results, but some clumps of tumour cells showed clear heterogeneity with regard to CD44 protein content. There were also some scattered focal discrepancies in the quantity and distribution of mRNA transcripts and proteins, respectively. CONCLUSIONS The ISH technique provides powerful independent corroboration of elevated CD44 gene expression and disproportionately high transcription of CD44v isoforms in carcinoma cells observed in earlier immunohistochemical and RT-PCR studies. It unequivocally localises the abnormally elevated gene transcription within the cancer cells and not in the surrounding inflammatory cells.