Aims: To characterize Gram-positive, catalase-negative, psychrotrophic, lactic acid-homo-fermentative, non-motile cocci isolated from vacuum-packaged refrigerated beef using phenotypic and genotypic methods. Methods and Results: A total of 89 strains was isolated at 2 and 6 weeks as one of the predominant microflora of five samples of vacuum-packaged beef stored at 2degreesC. The strains were compared with reference strains of some Gram-positive, catalase-negative cocci using SDS-PAGE whole-cell protein pattern analysis, biochemical characterization and 16S rDNA sequencing. The biochemical and physiological characteristics of the isolates resembled those of Lactococcus piscium GTC 552(T). Numerical analysis of the SDS-PAGE whole-cell protein patterns resulted in close clustering of the strains with L. piscium GTC 552(T) (r>0.68). Other Lactococcus and Leuconostoc species could be distinguished from the isolates using SDS-PAGE whole-cell protein patterns (r<0.58) and biochemical characteristics. The 16S rDNA sequencing of four randomly selected strains showed that the strains differed from L. piscium GTC 552(T) by two to three bases in the highly variable regions of the sequence. This is the first report on the isolation of L. piscium from vacuum-packaged beef. Conclusions: The Gram-positive catalase-negative cocci isolated from vacuum-packaged refrigerated beef have been identified as L. piscium. Significance and Impact of the Study: The findings of this work contribute to the knowledge of the microflora of vacuum-packaged refrigerated beef.