Abstractγ‐Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG‐tagged constructs, we have obtained evidence that γ‐tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ‐tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ‐Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ‐tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ‐tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time‐lapse imaging of photoconvertible protein Dendra2 tagged to γ‐tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ‐tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ‐tubulin and C53 was corroborated by pull‐down and co‐immunoprecipitation experiments. Overexpression of γ‐tubulin antagonized the inhibitory effect of C53 on DNA damage G2/M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ‐tubulin may also have nuclear‐specific function(s). J. Cell. Physiol. 227: 367–382, 2012. © 2011 Wiley Periodicals, Inc.