Ten Lactobacillus strains, previously isolated from different Bobal grape wines from the Utiel-Requena Origin Denomination of Spain, were characterized phylogenetically, genotypically and phenotypically. The 16S rRNA genes were sequenced and phylogenetic analysis showed that they form a tight phylogenetic clade that is closely related to reference strains Lactobacillus satsumensis NRIC 0604T, 'Lactobacillus uvarum' 8 and Lactobacillus mali DSM 20444T. DNA-DNA hybridization results confirmed the separation of the strains from other Lactobacillus species. Genotypically, the strains could be differentiated from their closest neighbours by 16S amplified rDNA restriction analysis and random amplified polymorphic DNA patterns. The strains were Gram-staining-positive, facultatively anaerobic rods that did not exhibit catalase activity. Phenotypically, they could be distinguished from their closest relatives by several traits such as their inabilities to grow at pH 3.3, to ferment sucrose, amygdalin and arbutin or to hydrolyse aesculin. The characteristics of the ten wine isolates suggest that they represent a novel species, for which the name Lactobacillus oeni sp. nov. is proposed. The type strain is 59bT (=CECT 7334T=DSM 19972T).