In normal tissues, the steady-state level of CD44 mRNA is low, and the variety of alternatively spliced transcripts produced by this complex gene is limited. Conversely, increased and disorderly expression of this gene has been observed in a number of types of cancer. This study analyzed the order in which the CD44 variant exons are spliced together in gastrointestinal tumor cell lines and in 20 colonic carcinomas and matched normal mucosa. We used a PCR-based assay to analyze specific exon junctions at the boundary of the standard and variant regions of the CD44 gene transcripts. This revealed characteristically different splicing preferences in colonic tumor and normal tissues. The junction of exon 5 to exon 8 appeared to be the most prevalent in normal mucosa, whereas the presence of junctions between exon 5 and either exon 7, 9, or 11 were increased markedly in tumor samples. These observations demonstrate that the unusual variety of CD44 transcripts in cancer cells results from the fidelity of alternative splicing mechanisms being compromised and are potentially useful as tumor cell markers in diagnostic assays.