Aims: The aim of this research was to characterize the metabolic pathway for maltose utilization in Enterococcus faecalis. Methods and Results: Screening a library of Enterococcus faecalis insertional mutants allowed the isolation of mutants affected in maltose utilization. Genetic analysis of the insertion loci revealed insertions in neighbour genes encoding an EII component of a phosphotransferase system (PTS) transporter (malT) and a maltose phosphorylase homologue (malP). The malP gene forms part of an operon which also includes genes encoding a phosphoglucomutase (malB), a mutarotase (aldose 1-epimerase) (malM) and a transcriptional regulator (malR). Analysis of 14C-labelled carbohydrates uptake revealed that more than 97% of maltose enters the cells by the PTS transporter MalT. Conclusions: Both experimental data and genetic organization of the malPBMR operon strongly suggest that in Enterococcus faecalis, maltose enters using a PTS, leaving maltose-6-phosphate inside the cells which is hydrolysed by a maltose phosphate phosphorylase (MalP). Significance and Impact of the Study: This study describes a new pathway for maltose utilization in lactic acid bacteria.