The centromere is a specialized chromosomal locus required for accurate chromosome segregation. A specific histone H3 variant, CENP-A, assembles at centromeres. CENP-A is required for kinetochore protein assembly and is an epigenetic marker for the maintenance of a functional centromere. Human CENP-A chromatin normally assembles on α-satellite DNA (alphoid DNA), a centromeric repetitive sequence. Using alphoid DNA arrays, human artificial chromosomes (HACs) have been constructed in human HT1080 cells and used to dissect the requirements for CENP-A assembly on DNA sequence. However, centromere formation is not a simple genetic event. In other commonly used human cell lines, such as HeLa and U2OS cells, no functional de novo centromere formation occurs efficiently with the same centromeric alphoid DNA sequences. Recent studies using protein tethering combined with the HAC system and/or genetic manipulation have revealed that epigenetic chromatin regulation mechanisms are also involved in the CENP-A chromatin assembly pathway and subsequent centromere/kinetochore formation. We summarize the DNA sequence requirements for CENP-A assembly and discuss the epigenetic regulation of human centromeres.