The isolation and localization of a chromosome 12-specific alpha satellite DNA sequence, p alpha 12H8, is described. This clone contains a complete copy of the 1.4-kb HindIII higher-order repeat present within the alpha satellite array on chromosome 12. The specificity of p alpha 12H8 was demonstrated by in situ hybridization and Southern blot analysis of a somatic cell hybrid mapping panel, both performed under high-stringency conditions. Polymorphic restriction patterns within the alpha satellite array, revealed by the use of the restriction enzymes BglII and EcoRV, were demonstrated to display Mendelian inheritance. These properties make p alpha 12H8 a valuable genetic marker for the centromeric region of chromosome 12.