We investigated the organization, architecture, and evolution of the largest cluster (∼4 Mb) of Krüppel-associated box zinc finger (KRAB-ZNF) genes located in cytogenetic band interval 19p12. A highly integrated physical map (∼700 kb) of overlapping cosmid and BAC clones was developed between genetic STS markers D19S454 and D19S269. Using ZNF91 exon-specific probes to interrogate a detailed EcoRI restriction map of the region, ZNF genes were found to be distributed in a head-to-tail fashion throughout the region with an average density of one ZNF duplicon every 150-180 kb of genomic distance. Sequence analysis of 208,967 bp of this region indicated the presence of two putative ZNF genes: one consisting of a novel member of this gene family (ZNF208) expressed ubiquitously in all tissues examined and the other representing a nonprocessed pseudogene (ZNF209), located 450 kb proximal to ZNF208. Large blocks of (∼25-kb) inverted ␤-satellite repeats with a remarkably symmetrical higher order repeat structure were found to bracket the functional ZNF gene. Hybridization analysis using the ␤-satellite repeat as a probe indicates that ␤-satellite interspersion between ZNF gene cassettes is a general property for 1.5 Mb of the ZNF gene cluster in 19p12. Both molecular clock data as well as a retroposon-mapping molecular fossil approach indicate that this ZNF cluster arose early during primate evolution (∼50 million years ago). We propose an evolutionary model in which heteromorphic pericentromeric repeat structures such as the ␤ satellites have been coopted to accommodate rapid expansion of a large gene family over a short period of evolutionary time.[The sequence data described in this paper have been submitted to GenBank under accession nos. AC003973 and AC004004.] Zinc finger (ZNF) genes represent one of the largest gene families in the human genome with an estimated 500-600 members (Hoovers et al. 1992;Becker et al. 1995;Klug and Schwabe 1995). Although the specific function of the majority of ZNF genes remains largely unknown, as a class they are believed to encode transcriptional regulators that in a few instances have been shown to play critical roles in cellular and developmental differentiation processes (Pieler and Bellefroid 1994). ZNF proteins have been implicated in many diverse eukaryotic developmental processes, such as segment pattern formation in the Drosophila embryo (Rosenberg et al. 1986); cellular proliferation in the cerebellar hindbrain of the mouse (Wilkinson et al. 1989); and hematopoietic differentiation among human myeloid precursor cells (Hromas et al. 1991). DNA binding of the encoded proteins is typically mediated by a ZNF motif that consists either of two cysteines and two histidines (Krüppel family or C 2 /H 2 type) or four cysteines alone (steroid receptor or C 2 / C 2 type). The conserved cysteines and/or histidines form a tetrahedral complex around a zinc metal ion, generating a folded loop or ''finger'' of 30 amino acids that is capable of making contact with DNA (Miller et al. 1985)...