The ade6-M26 mutation of Schizosaccharomyces pombe stimulates intragenic and intergenic meiotic recombination. M26 is a single base pair change creating a specific heptanucleotide sequence that is crucial for recombination hotspot activity. This sequence is recognized by proteins that may facilitate rate-limiting steps of recombination at the ade6 locus. To start the elucidation of the intermediate DNA structures formed during M26 recombination, we have analyzed the aberrant segregation patterns of two G to C transversion mutations flanking the heptanucleotide sequence in crosses homozygous for M26. At both sites the level of post-meiotic segregation is typical for G to C transversion mutations in S. pombe in general. Quantitative treatment of the data provides strong evidence for heteroduplex DNA being the major recombination intermediate at the M26 site. We can now exclude a double-strand gap repair mechanism to account for gene conversion across the recombination hotspot. Furthermore, the vast majority (> 95%) of the heteroduplexes covering either of the G to C transversion sites are produced by transfer of the transcribed DNA strand. These results are consistent with ade6-M26 creating an initiation site for gene conversion by the introduction of a single-strand or a double-strand break in its vicinity, followed by transfer of the transcribed DNA strands for heteroduplex DNA formation.