The position of the point mutation in the c-K-ras gene appears associated with different degrees of aggressiveness in human colorectal tumors. In addition, colon tumors carrying K-ras codon 12 mutations associate with lower levels of apoptosis than tumors lacking this mutation. To test the hypothesis of a distinct transforming capacity of different K-ras forms in an in vitro system, we generated stable transfectants of NIH3T3 cells expressing a plasmid containing K-ras mutated at codon 12 (K12) or at codon 13 (K13), or overexpressing the K-ras proto-oncogene (Kwt-oe). We evaluated changes in morphology, proliferative capacity, contact inhibition, and predisposition to apoptosis and anchorage-independent growth in K12, K13, and Kwt-oe transformants. In addition, we studied alterations in expression and/or activation of proteins that participate in signal transduction downstream of Ras or are involved in the regulation of apoptosis and cell-cell (E-cadherin and beta-catenin) and cell-substrate (focal adhesion kinase) interactions. We observed that K13 or Kwt-oe transformants died synchronically 24-48 h after reaching confluency. Their death was apoptotic. In contrast, K12 grew, forming bigger colonies with higher cell densities; and before reaching confluency, spontaneously formed spheroids and showed no sign of apoptosis. The enhanced resistance to apoptosis, loss of contact inhibition, and predisposition to anchorage-independent growth in the K12 transformants were associated with higher AKT/protein kinase B activation, bcl-2, E-cadherin, beta-catenin, and focal adhesion kinase overexpression, and RhoA underexpression, whereas the increased sensitivity of K13 or Kwt-oe transformants to apoptosis was associated with increased activation of the c-Jun-NH2-terminal kinase 1 pathway. All transformants showed a similar overactivation of mitogen-activated protein kinases and levels of bax expression similar to the endogenous level. Therefore, in our in vitro model, the localization of the mutation in the K-ras gene predisposes to a different level of aggressiveness in the transforming phenotype. K12 may increase aggressiveness not by altering proliferative pathways, but by the differential regulation of K-Ras downstream pathways that lead to inhibition of apoptosis, enhanced loss of contact inhibition, and increased predisposition to anchorage-independent growth. These results offer a molecular explanation for the increased aggressiveness of the tumors with K-ras codon 12 mutations observed in the clinical setting.