Oligopeptides are an important source of nutrients, but can serve also as signals for intercellular communication. Oligopeptide-binding proteins seem likely to play a role both in oligopeptide transport and in communication processes. One such protein, AmiA, has been identified in Streptococcus pneumoniae. amiA is the first gene of an operon, ami, which encodes a multicomponent oligopeptide transporter belonging to the family of ABC transporters (or traffic ATPases). This transporter was the first system of this type described in Gram-positive bacteria. To investigate the role and the subcellular location of the putative oligopeptide-binding protein in a bacterium devoid of periplasm, AmiA null mutants were first constructed. None was affected for oligopeptide uptake by the Ami system. Since this apparent dispensability of AmiA could result from a functional redundancy, we looked for chromosomal genes encoding homologues of AmiA. Two homologous genes were identified by DNA-DNA hybridization at low stringency with an amiA probe. Both genes (aliA and aliB) were cloned and shown to encode putative lipoproteins highly homologous to AmiA (close to 60% amino acid identity). Examination of all combinations of amiA, aliA and aliB mutations indicated that these proteins have overlapping specificities toward oligopeptides. The triple mutant is as deficient for oligopeptide transport as mutants in the amiCDE or F genes, which demonstrates that an oligopeptide-binding component is absolutely required for transport by the Ami system. Metabolic labelling with [3H]palmitic acid and cell fractionation were used to demonstrate that the three proteins are indeed membrane-bound lipoproteins in S. pneumoniae. This supports our previous hypothesis that substrate-binding lipoproteins are functionally equivalent to the periplasmic substrate-binding component of ABC transporters of Gram-negative bacteria. Finally, the observation that competence for genetic transformation was drastically reduced in a particular AliB mutant suggests that oligopeptide sensing is important for triggering competence.