In Bacillus transformation, sexual isolation is known to be an exponential function of the sequence divergence between donor and recipient. Here, we have investigated the mechanism under which sequence divergence results in sexual isolation. We tested the effect of mismatch repair by comparing a wild-type strain and an isogenic mismatch-repair mutant for the relationship between sexual isolation and sequence divergence. Mismatch repair was shown to contribute to sexual isolation but was responsible for only a small fraction of the sexual isolation observed. Another possible mechanism of sexual isolation is that more divergent recipient and donor DNA strands have greater difficulty forming a heteroduplex because a region of perfect identity between donor and recipient is required for initiation of the heteroduplex. A mathematical model showed that this heteroduplex-resistance mechanism yields an exponential relationship between sexual isolation and sequence divergence. Moreover, this model yields an estimate of the size of the region of perfect identity that is comparable to independent estimates for Escherichia coli. For these reasons, and because all other mechanisms of sexual isolation may be ruled out, we conclude that resistance to heteroduplex formation is predominantly responsible for the exponential relationship between sexual isolation and sequence divergence in Bacillus transformation.