The inheritance of chloroplast genes in Chlamydomonas is regulated by methylation of chloroplast DNA during gametogenesis. The wild-type pattern of maternal inheritance results from the methylation of chloroplast DNA in female (mt+) but not in male (mt-) gametes, leading to preferential degradation of chloroplast DNA of male origin in zygotes. This paper describes the distribution of 5-methyl cytosine residues in restriction fragments of chloroplast DNA sampled during gametogenesis by two methods: ethidium bromide staining of agarose gels, and binding of antibody directed against 5-methyl cytosine onto restriction fragments blotted to nitro-cellulose paper. Methylated cytosines are located in most if not all Eco RI and Msp I fragments, but the extent of methylation is not proportional to fragment size. The mat-1 mutation carried by males converts maternal inheritance. Chloroplast DNA of male gametes carrying the mat-1 mutation becomes methylated during gametogenesis. This methylation protects against restriction enzyme-promoted degradation in zygotes, as shown by physical data demonstrating the transmission to progeny of chloroplast genes carried on chloroplast DNA of the mat-1 male parent. Thus the mat-1 gene, which is linked to the mating-type locus, determines whether or not methylation of chloroplast DNA will occur in males during gametogenesis.