The ICF (immunodeficiency, centromeric instability and facial abnormalities) syndrome is a rare recessive disease characterized by immunodeficiency, extraordinary instability of certain heterochromatin regions and mutations in the gene encoding DNA methyltransferase 3B. In this syndrome, chromosomes 1 and 16 are demethylated in their centromere-adjacent (juxtacentromeric) heterochromatin, the same regions that are highly unstable in mitogen-treated ICF lymphocytes and B cell lines. We investigated the methylation abnormalities in CpG islands of B cell lines from four ICF patients and their unaffected parents. Genomic DNA digested with a CpG methylation-sensitive restriction enzyme was subjected to two-dimensional gel electrophoresis. Most of the restriction fragments were identical in the digests from the patients and controls, indicating that the methylation abnormality in ICF is restricted to a small portion of the genome. However, ICF DNA digests prominently displayed multicopy fragments absent in controls. We cloned and sequenced several of the affected DNA fragments and found that the non-satellite repeats D4Z4 and NBL2 were strongly hypomethylated in all four patients, as compared with their unaffected parents. The high degree of methylation of D4Z4 that we observed in normal cells may be related to the postulated role of this DNA repeat in position effect variegation in facio- scapulohumeral muscular dystrophy and might also pertain to abnormal gene expression in ICF. In addition, our finding of consistent hypomethylation and overexpression of NBL2 repeats in ICF samples suggests derangement of methylation-regulated expression of this sequence in the ICF syndrome.