Expression from a plasmid of the complete gene, including the codons for the N-terminal periplasmic targeting signal, for cytochrome c550 of Paracoccus denitrificans led to the formation of the holo protein in the periplasms of both P. denitrificans and Escherichia coli. Expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo-protein in the cytoplasms of both organisms. These findings are consistent with haem attachment occurring in the periplasm. In contrast, the formation of holo cytochrome c552 from Hydrogenobacter thermophilus following expression of the gene lacking the periplasmic targeting sequence in either P. denitrificans or E. coli is attributed to spontaneous cytoplasmic attachment of haem to the thermostable protein.