The mechanism of v-src-induced morphological transformation is still obscure. We compared LA29 rat fibroblasts, which express a temperature- sensitive (ts) v-src mutant, with D1025 rat fibroblasts, transfected with a ts mutant of v-fps. Upon transformation, LA29 cells adopted an elongated shape with reduced focal adhesions and less of actin stress fibers. In contrast, activation of v-fps in D1025 cells had little effect on morphology. In both cells, paxillin was strongly tyrosine phosphorylated upon activation of the kinases. This indicates that paxillin phosphorylation is not required, or not sufficient, for the v-src-induced disruption of focal adhesions. As previously described by others, v-src activated the ras-MAP kinase (MAPK) pathway, as indicated by tyrosine phosphorylation of the rasGAP-associated proteins p62 and p190 and MAPK phosphorylation. Since MAPK affects transcription, this suggested that novel gene transcription was required. This notion was confirmed using actinomycin D and cycloheximide, which did not impair activation of v-src kinase activity, but completely blocked v- src-induced morphological changes, as demonstrated using image analysis. Furthermore, we observed that v-src-induced changes in cell shape occurred before the reduction in number and size of focal adhesions. We conclude that v-src-induced transformation of rat fibroblasts depends on synthesis of a protein, which induces rapid changes in cell shape that precede the loss of focal adhesions.