beta satellite is a repetitive DNA family that consists of approximately 68-bp monomers tandemly repeated in arrays of at least several hundred kilobases. In this report we describe and characterize two subfamilies located exclusively on the human acrocentric chromosomes. The first subfamily is defined by a homogeneous approximately 2.0-kb higher-order repeat unit and is located primarily distal to the ribosomal RNA gene cluster, based both on fluorescence in situ hybridization to metaphase chromosomes and on filter hybridization analysis of translocation chromosomes isolated in somatic cell hybrids. In contrast, the second subfamily is located both distal and proximal to the ribosomal RNA gene cluster on the same acrocentric chromosomes. The DNA sequences of a number of monomers from these two subfamilies are compared to each other and to other beta satellite monomers to assess both inter- and intrasubfamily sequence relationships for these monomers.