We recently showed that rap1 regulates growth and proliferation in normal keratinocytes, which provoked us to investigate its expression and regulation in malignant cells. Rap1 is variably expressed in whole cell lysates of squamous cell carcinoma (SCC) cell lines. Immunoblot analysis of nuclear and cytosolic fractions and immunohistochemistry revealed that in addition to cytoplasmic expression, SCC cells also exhibit prominent punctate rap1 expression in the nucleus. This unexpected nuclear distribution was confirmed by the evaluation of human oral cancer specimens by immunohistochemistry, which showed both nuclear and cytoplasmic localization. Cytoplasmic rap1 expression was observed mostly in large differentiated cells, whereas nuclear localization was found in morphologically less differentiated cells. Quantitative reverse transcriptase polymerase chain reaction and Northern blot analysis showed that both rap1A and rap1B are expressed in SCC cell lines although rap1B signals are more prominent. Transfection with enhanced GFP-tagged constitutively active and inactive forms of rap1B demonstrated that the active GTP-bound form translocates to the nucleus whereas inactive rap1B(GDP) is retained in the cytoplasm, much of which is in a perinuclear distribution. Furthermore, growth factors induce nuclear translocation of rap1 in oral cancer cells. This novel discovery that active, GTP-bound rap1 translocates to the nucleus makes it only the second of over 100 small GTP-binding proteins to be identified in the nucleus, and the striking prominence of rap1 expression in the nucleus of SCC cells suggests that activated rap1 plays a role in the malignant process.