Although RhoA plays an important role in cell proliferation and in Ras transformation in fibroblasts and mammary epithelial cells, its role in intestinal epithelial cells is unknown. In a previous study, we showed that polyamine depletion (DL-alpha-difluoromethylornithine, DFMO, treatment) strongly inhibits the proliferation of intestinal epithelial cells (IEC-6). In this report, we examined the effect of RhoA on IEC-6 cell proliferation and whether polyamine depletion inhibits cell proliferation in the presence of constitutively active RhoA. Constitutively active RhoA and vector transfected IEC-6 cell lines were grown in the presence or absence of DFMO, which causes polyamine depletion by inhibiting ornithine decarboxylase (ODC), the first rate-limiting step in polyamine synthesis. Constitutively active RhoA significantly increased the rate of cell proliferation. These cells also lost contact inhibition and formed conspicuous foci when they were fully confluent. Decreased p21Waf1/Cip1 expression, increased Cdk2 mRNA levels and activity accompanied the increased proliferation. The inhibition of p21^Waf1/Cip1 was independent of p53. There was no activation of the Ras-Raf-MEK-ERK pathway in the RhoA transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on IEC-6 cell proliferation, focus formation, and Cdk2 expression. The stability of mRNA and protein for Cdk2 and p21^Waf1/Cip1 in V14-RhoA cells was not significantly different from that of vector transfected cells. In conclusion, RhoA activation decreased p21^Waf1/Cip1 expression, increased basal and serum-induced ODC activity, Cdk2 expression, Cdk2 protein and Cdk2 activity, leading to the stimulation of intestinal epithelial cell proliferation and transformation. Polyamine depletion totally prevented RhoA's effect on proliferation by decreasing Cdk2 expression and activity.