Although the KRAB zinc finger proteins probably constitute the single largest class of transcription factors within the human genome, almost nothing is known about their biological function. To increase our knowledge about this interesting and relatively unexplored family of potent transcriptional repressors, we here present the cloning, structural analysis, and expression study of three novel mouse KRAB zinc finger proteins. In addition, we present an extensive comparative analysis of various members of this gene family based on the structure of the common KRAB A motif. At least three larger subfamilies of KRAB zinc finger proteins are identified: one carrying the classical KRAB A motif only, another holding both a classical KRAB A and a classical KRAB B motif, and a third holding a classical KRAB A and a highly divergent KRAB B domain, named b. A large variation both in size and in primary amino acid sequence was observed in the linker region between the KRAB domain and the C-terminally located zinc finger repeats. This variability indicates that this region is of minor importance for the biological function of KRAB-containing zinc finger proteins. The fact that in many zinc finger genes, the entire or almost the entire linker region is composed of degenerate finger motifs substantiates this conclusion. The absence of identifiable KRAB A and B motifs in the genome of yeast, Saccharomyces cerevisiae, indicates a relatively late appearance of the KRAB domain in evolution and may suggest that the biological functions are restricted to multicellular organisms. In addition, we show that the expression of individual members of one subfamily of KRAB zinc finger genes is restricted to specific hematopoietic cell lineages. This finding suggests that KRAB zinc finger proteins may play a role in lineage commitment, possibly silencing leakage transcription from nonlineage-expressed genes.