Small GTPases of the Rho family are involved in the regulation of a variety of cellular processes, such as the organization of the microfilamental network, cell-cell contact and malignant transformation. To address the question of whether Rho proteins are involved in carcinogenesis in man, we compared their expression in tumors from colon, breast and lung with that of the corresponding normal tissue originating from the same patient. As shown by Rho-specific 32P-ADP-ribosylation, as well as Western-blot analysis, the amount of RhoA protein was largely increased in all 3 types of tumors tested. The most dramatic differences in the expression of Rho GTPases were observed in breast tissue. All breast tumors analyzed showed high levels of RhoA, Rac and Cdc42 proteins, whereas in the corresponding normal tissue these Rho proteins were hardly or not detectable. Progression of breast tumors from WHO grade I to grade III was accompanied by a significant average increase in RhoA protein. Overall, increase in the amount of Rho GTPases, in particular RhoA, appears to be a frequent event in different types of human tumors. This supports the view that Rho GTPases are involved in human carcinogenesis. Int. J. Cancer 81:682–687, 1999. © 1999 Wiley-Liss, Inc.