An experimental system developed previously for the heterologous expression of c-type cytochromes in Escherichia coli Q1has been adapted to monitor protein transfer across the bacteria's cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec-specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat-mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system.