The delta pH-driven and Sec-related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH-dependent system contain a common twin-arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH-driven translocation process; substitution of the arg-arg by gln-gln or even arg-lys totally blocks translocation across the thylakoid membrane, and replacement by lys-arg reduces the rate of translocation by > 100-fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin-arg motif into a Sec-dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec-type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.