GC clusters constitute the major repetitive elements in the mitochondrial (mt) genome of the yeast Saccharomyces cerevisiae. Many of these clusters are optional and thus contribute much to the polymorphism of yeast mtDNAs. We have made a systematic search for polymorphic sites by comparing mtDNA sequences of various yeast strains. Most of the 26 di- or polymorphic sites found differ by the presence or absence of a GC cluster of the majority class, here referred to as the M class, which terminate with an AGGAG motif. Comparison of sequences with and without the GC clusters reveal that elements of the subclasses M1 and M2 are inserted 3' to a TAG, flanked by A + T rich sequences. M3 elements, in contrast, only occur in tandem arrays of two to four GC clusters; they are consistently inserted 3' to the AGGAG terminal sequence of a preexisting cluster. The TAG or the terminal AGGAG, therefore, are regarded as being part of the target sites for M1 and M2 or M3 elements, respectively. The dinucleotide AG is in common to both target sites; it also occurs at the 3' terminus (AGGAG). This suggests its duplication during GC cluster insertion. This notion is supported by the observation that GC clusters of the minor classes G and V similarily repeat at their 3' terminus a GT or an AA dinucleotide, respectively, from their putative target sites.