Altered genomic methylcytosine content has been described for a number of tumor types, including neuroblastoma. However, it remains to be determined for different tumor types whether specific loci or chromosomal regions are affected by a methylation change or whether the change is random. We have implemented a computer-based approach for the analysis of two-dimensional separations of human genomic restriction fragments. Through the use of methylation-sensitive restriction enzymes, methylation differences in genomic DNA between tumor and normal tissues can be detected. We report the cloning and sequencing of two fragments detectable in two-dimensional separations of genomic DNA of neuroblastomas. These fragments were found to be a part of repetitive units that exhibited demethylation in neuroblastoma relative to other tumor types. Our finding of a distinct pattern of methylation of repetitive units in neuroblastoma suggests that altered methylation at certain loci may contribute to the biology of this tumor.