Here we demonstrate that endogenous human homeodomain-interacting protein kinase (HIPK) 2 and the highly homologous kinase HIPK3 are found in a novel subnuclear domain, the HIPK domains. These are distinct from other subnuclear structures such as Cajal bodies and nucleoli and show only a partial colocalization with promyelocytic leukemia (PML) nuclear bodies (PML-NBs). A kinase inactive HIPK2 point mutant is localized in the nucleoplasm. The occurrence of HIPK domains in PML^−/− fibroblasts reveals their independence from the PML protein. HIPK2 can be almost completely recruited to PML-NBs by the PML isoform PML IV, but not by PML-III. PML IV-mediated recruitment of HIPK2 does not rely on its kinase function and also occurs in PML^−/− fibroblasts, showing that this PML isoform is sufficient for recruitment of HIPK2. Whereas the architecture of HIPK domains is PML independent, HIPK2-mediated enhancement of p53-dependent transcription, p53 serine 46 phosphorylation and the antiproliferative function of HIPK2 strictly rely on the presence of PML.