We have described previously the use of microarray technology to identify novel target genes of TGF-beta (transforming growth factor-beta) signalling in mouse embryo fibroblasts deficient in Smad2 or Smad3 [Yang, Piek, Zavadil, Liang, Xie, Heyer, Pavlidis, Kucherlapati, Roberts and Böttinger (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 10269-10274]. Among the TGF-beta target genes identified, a novel gene with sequence homology to members of the Ras superfamily was identified, which we have designated as RLP (Ras-like protein). RLP is a Smad3-dependent immediate-early TGF-beta target gene, its expression being induced within 45 min. Bone morphogenetic proteins also induce expression of RLP, whereas epidermal growth factor and phorbol ester PMA suppress TGF-beta-induced expression of RLP. Northern-blot analysis revealed that RLP was strongly expressed in heart, brain and kidney, and below the detection level in spleen and skeletal muscles. At the protein level, RLP is approx. 30% homologous with members of the Ras superfamily, particularly in domains characteristic for small GTPases. However, compared with prototypic Ras, RLP contains a modified P-loop, lacks the consensus G2 loop and the C-terminal prenylation site and harbours amino acid substitutions at positions that render prototypic Ras oncogenic. However, RLP does not have transforming activity, does not affect phosphorylation of mitogen-activated protein kinase and is unable to bind GTP or GDP. RLP was found to associate with certain subtypes of the TGF-beta receptor family, raising the possibility that RLP plays a role in TGF-beta signal transduction. Although RLP did not interact with Smads and did not affect TGF-beta receptor-induced Smad2 phosphorylation, it inhibited TGF-beta-induced transcriptional reporter activation, suggesting that it is a novel negative regulator of TGF-beta signalling.