Inactivation of the murine TATA binding protein (TBP) gene by homologous recombination leads to growth arrest and apoptosis at the embryonic blastocyst stage. However, after loss of TBP, RNA polymerase II (pol II) remains in a transcriptionally active phosphorylation state, and in situ run-on experiments showed high levels of pol II transcription comparable to those of wild-type cells. In contrast, pol I and pol III transcription was arrested. Our results show a differential dependency of the RNA polymerases on TBP and provide evidence for TBP-independent pol II transcriptional mechanisms that allow reinitiation and maintenance of gene transcription in vivo.