Long expansions of transcribed trinucleotide microsatellites have been etiologically associated with some neurological diseases. The investigation of such novel polymorphisms has thus become a subject of great interest. We searched the expressed sequence tag databank for reiterated trinucleotides and selected EST00493 (D2S196E) with 14 tandem ACA triplets as a potentially polymorphic locus. Size variation was readily detected, with four common alleles containing 12–15 repeats. In addition, we observed distinct heteroduplexes in amplifications from individuals with identical ACA genotypes. Sequencing of their polymerase chain reaction (PCR) products revealed a G→A transition immediately preceding the trinucleotide repeats, hence defining 8 distinct haplotypes and 36 possible genotypes. Indeed, mutation detection enhancement gel electrophoresis of mixed PCR products from cloned haplotypes revealed 24 distinct heteroduplex patterns for the six possible trinucleotide heterozygotes. The observation of heteroduplex patterns in non-denaturing polyacrylamide gel electrophoresis (instead of the more commonly used denaturing gels) can thus be utilized to increase the informativeness of microsatellite polymorphisms by unraveling otherwise cryptic sequence variation. The D2S196E polymorphism has proved useful for demonstrating microsatellite instability and loss of heterozygosity in colorectal tumors.