Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated am RIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.Certain cytosines in the DNA of many animals, plants, and fungi are methylated. The importance of DNA methylation in mice is highlighted by the finding that it is essential for embryogenesis (24). Many clues to the function(s) of methylation are emerging. DNA methylation has been implicated in X-chromosome inactivation (49), genomic imprinting (4, 38), and other processes. Methylation of promoter sequences typically correlates with gene inactivity (1); however, it is unclear to what extent methylation is used by eukaryotic organisms to regulate transcription.Despite progress made in understanding functions of DNA methylation, little is known about the control of methylation in eukaryotes. In mammals, DNA methylation patterns are reorganized during gametogenesis and embryogenesis (28), but the patterns are relatively stable after differentiation of tissues. 5-Methylcytosine (m 5 C) is predominantly located within CpG dinucleotides in animals (50). Riggs (36) and Holliday and Pugh (20) proposed that a ''maintenance methylase'' propagates methylation patterns by acting on hemimethylated 5Ј-CpG-3Ј/GpC dinucleotides in newly replicated DNA. Aspects of this model have been supported, but non-CpG methylation (54, 60) and methylation heterogeneity evidenced by partially methylated sites (56, 63) challenge the model (39). How methylation patterns are first established also remains largely a mystery, although results of recent studies in mammals suggest that binding of the transcription factor Sp1 can keep regions unmethylated (5, 25).The fungus Neurospora crassa provides an excellent system to study DNA methylation. Most of the genome appears devoid of methylation; however, several densely methylated endogenous...