Krüppel-related zinc finger proteins, with 564 members in the human genome, probably constitute the largest individual family of transcription factors in mammals. Approximately 30% of these proteins carry a potent repressor domain called the Krüppel associated box (KRAB). Depending on the structure of the KRAB domain, these proteins have been further divided into three subfamilies (A + B, A + b, and A only). In addition, some KRAB zinc finger proteins contain another conserved motif called SCAN. To study their molecular evolution, an extensive comparative analysis of a large panel of KRAB zinc finger genes was performed. The results show that both the KRAB A + b and the KRAB A subfamilies have their origin in a single member or a few closely related members of the KRAB A + B family. The KRAB A + B family is also the most prevalent among the KRAB zinc finger genes. Furthermore, we show that internal duplications of individual zinc finger motifs or blocks of several zinc finger motifs have occurred quite frequently within this gene family. However, zinc finger motifs are also frequently lost from the open reading frame, either by functional inactivation by point mutations or by the introduction of a stop codon. The introduction of a stop codon causes the exclusion of part of the zinc finger region from the coding region and the formation of graveyards of degenerate zinc finger motifs in the 3'-untranslated region of these genes. Earlier reports have shown that duplications of zinc finger genes commonly occur throughout evolution. We show that there is a relatively low degree of sequence conservation of the zinc finger motifs after these duplications. In many cases this may cause altered binding specificities of the transcription factors encoded by these genes. The repetitive nature of the zinc finger region and the structural flexibility within the zinc finger motif make these proteins highly adaptable. These factors may have been of major importance for their massive expansion in both number and complexity during metazoan evolution.