We used a proteomic approach to identify constitutively formed extracellular proteins of Hypocrea atroviridis (Trichoderma atroviride), a known biocontrol agent. The fungus was cultivated on glucose and the secretome was examined by two-dimensional gel electrophoresis. The two predominant spots were identified by MALDI MS utilizing peptide mass fingerprints and amino acid sequence tags obtained by postsource decay and/or high-energy collision-induced dissociation (MS/MS) experiments, and turned out to be the same protein (12 629 Da as determined with MS, pI 5.5–5.7), probably representing the monomer and the dimer. The corresponding gene was subsequently cloned from H. atroviridis and named epl1 (eliciting plant response-like), because it encodes a protein that exhibits high similarity to the cerato-platanin family, which comprises proteins such as cerato-platanin from Ceratocystis fimbriata f. sp. platani and Snodprot1 of Phaeosphaeria nodorum, which have been reported to be involved in plant pathogenesis and elicitation of plant defense responses. Additionally, based on the similarity of the N-terminus to that of H. atroviridis Epl1, we conclude that a previously identified 18 kDa plant response elicitor isolated from T. virens is an ortholog of epl1. Our results showed that epl1 transcript was present under all growth conditions tested, which included the carbon sources glucose, glycerol, l-arabinose, d-xylose, colloidal chitin and cell walls of the plant pathogen Rhizoctonia solani, and also plate confrontation assays with R. solani. Epl1 transcript could even be detected under osmotic stress, and carbon and nitrogen starvation.