Homologous recombination between dispersed DNA repeats creates chromosomal rearrangements that are deleterious to the genome. The methylation associated with DNA repeats in many eukaryotes might serve to inhibit homologous recombination and play a role in preserving genome integrity. We have tested the hypothesis that DNA methylation suppresses meiotic recombination in the fungus Ascobolus immersus. The natural process of methylation-induced premeiotically (MIP) was used to methylate the b2 spore color gene, a 7.5-kb chromosomal recombination hot spot. The frequency of crossing-over between two markers flanking b2 was reduced several hundredfold when b2 was methylated on the two homologs. This demonstrates that DNA methylation strongly inhibits homologous recombination. When b2 was methylated on one homolog only, crossing-over was still reduced 50-fold, indicating that the effect of methylation cannot be limited to the blocking of initiation of recombination on the methylated homolog. On the basis of these and other observations, we propose that DNA methylation perturbs pairing between the two intact homologs before recombination initiation and/or impairs the normal processing of recombination intermediates.[Key Words: Ascobolus immersus; chromosome rearrangements; crossing-over; DNA methylation; meiotic recombination; repeat DNA sequences] Received November 28, 1997; revised version accepted February 20, 1998. Crossing-over between dispersed DNA repeats results in chromosomal rearrangements. In eukaryotes, the destructive potential of dispersed repeats through ectopic homologous recombination is well documented (Rouyer et al. 1987;Montgomery et al. 1991;Small et al. 1997). In yeast, artificial duplications placed in ectopic position can interact and generate chromosomal rearrangements through homologous recombination at high frequency during meiosis (Lichten et al. 1987). In higher eukaryotes, the number of repeats per genome is often so high that no single cell would escape genomic rearrangements if ectopic recombination were to occur at high frequencies. Therefore, factors must exist that limit recombination between dispersed repeats. Thuriaux (1977) pointed out that the frequency of crossing-over per unit of physical DNA length decreases with increasing genome size, and proposed that recombination is confined to genes. According to this hypothesis, satellite DNA sequences and interspersed DNA repeats, which constitute the bulk of the intergenic regions, must be poor substrates for meiotic recombination even when in allelic positions. Nucleotidic divergence (Rayssiguier et al. 1989;Radman and Wagner 1993) and an insufficient length of sequence identity (Shen and Huang 1986;Jinks-Robertson et al. 1993) are two factors known to reduce drastically homologous recombination. Nevertheless, other factors that suppress homologous recombination are required to protect genomes against the threat generated by families of long DNA repeats that have diverged little or not at all (e.g., after recent duplication events)....