The inactivation of one of the two X chromosomes in females is a random process associated with methylation principally in CpG islands. The methylation status of a CpG island in intron 22 of the human factor VIII (FVIII) gene was investigated using a novel practical approach. Genomic DNA from men and women was digested with various methylation-sensitive (MS) restriction enzymes, the recognition sequences of which occurred at least once in the FVIII CpG island. Long distance-polymerase chain reaction (LD-PCR) was then used to amplify the island. Successful amplification indicated that the island was methylated and the absence of a PCR product indicated that at least one restriction site was unmethylated. To analyze the relative methylation status of the extragenic and intragenic copies of the island, we used Southern blot with MS restriction enzymes. The MS LD-PCR patterns obtained from male and female DNA samples indicated that at least some copies of the intragenic CG island were fully methylated at all sites investigated. Additionally, the island showed consistent differences among individuals. Southern blot studies using female DNA showed partial resistance to MS digestion for the intragenic and extragenic CpG island homologs. Our observations indicate that this CpG island is predominantly methylated on the X chromosome of males and suggest that its methylation pattern does not correlate with X inactivation of females. This prevents the use of this island coupled with DNA polymorphisms for investigation of X-chromosome inactivation.