The p21 proteins encoded by N-, Ki-, and H-ras are small guanine nucleotide-binding proteins that act as switches in several signal transduction pathways. Recently, evidence has been accumulating to suggest that valine-12 mutation in the Ki-Ras protein is associated with lung and colorectal tumours that are more aggressive than those carrying aspartate-12 mutation. The purpose of this study was to determine whether cells transfected with different Ki-ras codon-12 mutants have different biological behaviours in vitro that could reflect the differences in behaviour in vivo. For that reason, Rat-1 fibroblasts transfected with the valine-12 or aspartate-12 mutant or the wild-type Ki-ras gene were assessed in terms of in vitro invasion, transformation, and VEGF production. Both mutants demonstrated equal abilities to transform Rat-1 cells and induce VEGF production, while cells transfected with wild-type Ki-Ras failed to do so. Most significantly, the valine-12 mutants demonstrated a greater ability to invade Matrigel than cells expressing the aspartate-12 mutant or wild-type Ki-Ras proteins. This study complements previous experimental data that specific Ras mutations differ in their effects in vivo and shows, for the first time, a significant difference in Matrigel invasion in vitro. The precise mechanisms behind these biological differences in vivo and in vitro should now be investigated.