Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF-β1 (transforming growth factor-β1) and small G protein RhoA in tumour progression, the influence of TGF-β1 treatment or RhoA-associated kinase inhibitor on the production of NO (nitric oxide) and MMP-2 and MMP-9 (metalloproteinases-2 and -9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP-2 and MMP-9. rhTGF-β1 and the synthetic Rho kinase inhibitor (Y-27632) decreased MMP-2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF-β1 decreased NO secretion by cells, while Y-27632 had no effect on it. Immunoblotting with anti-RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF-β1. rhTGF-β1 induced α-smooth muscle actin (α-SMA) expression, especially in high Duke's grade of colon cancer, while Y-27632 blocked it. Summing up, in colon carcinoma cells, TGF-β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α-SMA expression or assayed using motility data and may be a good target for cancer therapy.