We have determined the orientation of the major centromeric alphoid array on the human Y chromosome. A PCR assay was used to analyse the vector-insert junctions of seven YAC clones previously positioned on two independent Y chromosomes. The orientation is the same at all 10 positions measured. This suggests that the alphoid array is a simple unidirectional repeat throughout and that human centromere structure thus differs from the palindromic organisation found in Schizosaccharomyces pombe.