The twin arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. In Gram-negative bacteria, membrane-bound TatABC subunits are all essential for activity, whereas Gram-positive bacteria usually contain only TatAC subunits. In Bacillus subtilis, two TatAC-type systems, TatAdCd and TatAyCy, operate in parallel with different substrate specificities. Here, we show that they recognize similar signal peptide determinants. Both systems translocate green fluorescent protein fused to three distinct Escherichia coli Tat signal peptides, namely DmsA, AmiA and MdoD, and mutagenesis of the DmsA signal peptide confirmed that both Tat pathways recognize similar targeting determinants within Tat signals. Although another E. coli Tat substrate, trimethylamine N-oxide reductase, was translocated by TatAdCd but not by TatAyCy, we conclude that these systems are not predisposed to recognize only specific Tat signal peptides, as suggested by their narrow substrate specificities in B. subtilis. We also analysed complexes involved in the second Tat pathway in B. subtilis, TatAyCy. This revealed a discrete TatAyCy complex together with a separate, homogeneous, approximately 200 kDa TatAy complex. The latter complex differs significantly from the corresponding E. coli TatA complexes, pointing to major structural differences between Tat complexes from Gram-negative and Gram-positive organisms. Like TatAd, TatAy is also detectable in the form of massive cytosolic complexes.