This study characterizes the S-6m-haplotype, a mutated S-6-haplotype with an altered HindIII cut site, of sour cherry (Prunus cerasus). Inheritance and pollination studies of S-haplotypes from reciprocal crosses between 'Erdi Botermo' (EB; (SSSa)-S-4-S-6m) and 'Rheinische Schattenmorelle' (RS; (SSSSc)-S-6-S-a-S-b) revealed that the S-6m-haplotype conferred unilateral incompatibility with a non-functional pistil component and a functional pollen component. Expression analyses of S-6-RNase and SFB6, a candidate gene for pollen-S, in the S-6m-haplotype showed that SFB6 was transcribed in EB pollen, but S-6-RNase was not transcribed in EB styles. These results were consistent with data from the inheritance and pollination studies. Inverse PCR for the flanking regions of S-6-RNase in the S-6- and S-6m-haplotypes revealed an approximately 2600 bp insertion present at approximately 800 bp upstream of the S-6-RNase in the S-6m-haplotype, which is responsible for the alternation of the HindIII cut site and a possible cause of inhibition of the transcription of S-6-RNase. SFB6 was present downstream of S-6-RNase in both the S-6- and S-6m-haplotypes and expressed in the same way, supporting the idea that SFB is a good candidate for pollen-S in Prunus.