The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial cytoplasmic membranes. The Tat system of Streptomyces lividans consists of TatA, TatB and TatC, unlike most Gram-positive bacteria, which only have TatA and TatC subunits. Interestingly, in S. lividans TatA and TatB are localized in both the cytoplasm and the membrane. In the cytoplasm soluble TatA and TatB were found as monomers or as part of a hetero-oligomeric complex. Further analysis showed that specific information for recognition of the precursor by the soluble Tat components is mainly present in the twin-arginine signal peptide. Study of the role of the Tat subunits in complex assembly and stability in the membrane and cytoplasm showed that TatB stabilizes TatC whereas a key role in driving Tat complex assembly is suggested for TatC. Finally, by analysis of the oligomeric properties of TatA in the membrane of S. lividans and study of the affinity of membrane-embedded TatA for Tat/Sec precursors, a role for TatA as a translocator is postulated.