Immunolocalization and siRNAi cell fractionation plus expression of FLAG-tagged constructs showed presence of γ-tubulin in nucleoli of diverse mammalian interphase cells. It was found outside fibrillar centers of ribosomal DNA transcription, associated with nucleolar remnants after nuclear envelope breakdown and translocated to nucleoli during mitosis. Leptomycin B cell pretreatment did not affect nuclear γ-tubulin distribution, making unlikely rapid active transport of γ-tubulin via nuclear pores into the nucleus. This was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Further experiments revealed and confirmed association of γ-tubulin with tumor suppressor protein C53 located in many subcellular compartments including nucleoli. γ-Tubulin overexpression overcame C53 block of DNA damage G(2)/M checkpoint activation. Beside its known role in microtubule nucleation γ-tubulin may thus also have nuclear-specific function(s).