The protozoan intestinal parasite Giardia lamblia lacks mitochondria and the ability to make haem yet encodes several putative haem-binding proteins, including three of the cytochrome b(5) family. We cloned one of these (gCYTb5-I) and expressed it within Escherichia coli as a soluble holoprotein. UV-visible and resonance Raman spectra of gCYTb5-I resemble those of microsomal cytochrome b(5), and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the haem iron. The reduction potential of gCYTb5-I is -165 mV vs. SHE and is relatively low compared to most values (-110 to +80 mV) for this class of protein. The amino- and carboxy-terminal sequences that flank the central haem-binding core of the Giardia cytochromes are highly charged and differ from those of other family members. A core gCYTb5-I variant lacking these flanking sequences was also able to bind haem. The presence of one actual and two probable functional cytochromes b(5) in Giardia is evidence of uncharacterized cytochrome-mediated metabolic processes within this medically important protist.