The alphoid DNA family is composed of tandemly repeated sequences whose organization is chromosome specific. Under stringent conditions of hybridization, subsets of these sequences localize specifically to the centromeric region of a given chromosome. The alphoid clone, 308 (D6Z1), is a 3-kb BamHI DNA fragment that is repeated at the centromere of chromosome 6. Sequences homologous to 308 are organized as 2-kb BamHI repeats on X. We used 308 to screen a 2-kb BamHI genomic library and obtained the cloned homologue, XC, which hybridizes, in situ, to the centromere of X. These probes provide a means of analyzing the centromeric region on two different human chromosomes. The complete nucleotide sequence of 308 shows that it is composed of 20 variant 173-bp repeats. The organization of the 173-bp monomers is not a repetitious but a symmetric pattern, and an inversion is present. The chromosome specificity of 308 to chromosome 6 is determined by the entire 3-kb sequence and not by an amplified 173-bp monomer, because the divergence among the 173-bp units on chromosome 6 is as great as that between monomers on chromosomes 6 and X. Therefore, the organization of monomers is specific for a chromosome, and the monomers themselves are not specific. Analysis of the sequence data from these two alphoid sequences and other published sequences shows that some chromosomes have sequences that are more homologous than others. These subsets of alphoid sequences reflect the latters' chromosomal distribution and evolution. By using probes 308 and XC, we found that alphoid repeats from specific chromosomes are similar in that there is no evidence for their transcription in lymphoblasts and fibroblasts, and we demonstrated the possibility that all of these sequences are late replicating.