The distribution of the modified base 5-methylcytosine in nuclear DNA from the slime mould Physarum polycephalum was examined using the methylation-sensitive restriction endonucleases Hpa II and Hha I. Using these enzymes, Physarum DNA can be separated into high (M+) and low (M-) molecular weight fractions comprising 20% and 80% of the genome respectively. The M+ fraction is reduced in size by the methylation-insensitive isoschizomer of Hpa II, Msp I, thus showing that C-C-G-G sites modified to C-meC-G-G are present in this fraction. In addition, it was shown that C-C-G-G sites modified as meC-C-G-G are also present in Physarum DNA. Base composition analyses using HPLC showed that approximately 7% of all cytosines are methylated, and that of these 70% are located in the M+ fraction. Calculations suggest that 5-meC residues probably occur in cytosine-containing dinucleotides other than CpG. The distribution of repetitive DNA sequence families between the M+ and M- fractions was investigated both by restriction analysis of purified M+ DNA, and by hybridization analysis using specific cloned Physarum DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both fractions, although some repetitive sequences are enriched either in M+ or in M- DNA. The results of the hybridization analysis also indicate that C-meC-G-G and meC-C-G-G sites occur along the same DNA sequence tracts in the genome. Attempts to study the sequence organisation of the M+ fraction by cloning large segments of Physarum DNA were unsuccessful, since cosmid or phage lambda clones containing large Physarum DNA inserts are unstable and suffer substantial deletions. However, cosmid or phage lambda clones constructed using Trypanosoma brucei DNA are perfectly stable. Thus, it is concluded that the instability of the Physarum DNA clones is a result of some property of the Physarum DNA, and not an artefact of the cloning procedures used.