Activated RAS promotes dimerization of members of the RAF kinase family . ATP-competitive RAF inhibitors activate ERK signalling 4-7 by transactivating RAF dimers . In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumourspecific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E) 8 . However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.RAF inhibitors have remarkable clinical activity in mutant BRAF melanomas that is limited by acquisition of drug resistance 8 . To identify new mechanisms of resistance, we generated cell lines resistant to vemurafenib by exposing the BRAF-mutant (V600E) melanoma cell line SKMEL-239 to a high dose of drug (2 mM). At this concentration, vemurafenib effectively inhibited ERK signalling and induced cell cycle arrest and cell death (Fig. , Supplementary Fig. and data not shown). Five independent vemurafenib-resistant cell populations were generated after approximately 2 months of continuous drug exposure (Fig. ). We chose this approach rather than one of gradual adaptation to increasing concentrations of drug because it more closely represents the clinical situation 8 .Resistance of SKMEL-239 cells to vemurafenib was associated with decreased sensitivity of ERK signalling to the drug (Fig. and Supplementary Fig. ). Analysis revealed the presence of two distinct classes of resistant clones. In the first, exemplified by the C3 clone, the half-maximum inhibitory concentration (IC 50 ) for phosphorylated MEK (pMEK) inhibition was more than 100-fold higher than that of