The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity towards sucrose was approximately 10 times higher than towards raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-micron plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii. ; Project AshByofactory (grant PTDC/EBB-EBI/101985/2008 - FCOMP-01-0124-FEDER-009701), MIT-Portugal Program (PhD grant SFRH/BD/39112/2007 to Tatiana Q. Aguiar) and grant SFRH/BDP/63831/2009 to Carla Oliveira.