Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH 2 -terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1 transactivating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expressing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1 trans-activating activity are not absolute requirements for ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the proteasedependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype. These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. In ras-transformed NIH 3T3 fibroblasts, the uPAand CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.The Ras protein is at the center of a web of multiple downstream pathways that are differentially regulated to produce signals that control processes as disparate as cell division, cell differentiation, and programmed cell death (reviewed in Refs. 1-3). Two of the better characterized pathways downstream from Ras are from Ras to ERK-1 1 and -2 through the sequential activation of Raf and MEK1, and the pathway from Ras leading to the activation of JNK1 through the activation of Cdc42 and Rac 1 which then sequentially activate PAK, MEK kinase-2 and/or -3, JNKK/SEK/MKK4, and JNK1. The ERKs and JNK1 are mitogen-activated protein kinases that translocate to the nucleus and activate transcription factors involved in Rasregulated processes. The protein c-Jun is a nuclear substrate of JNK1 (4 -7), and c-Jun is a prominent component of many AP-1 transcription factors that ...