One characteristic of pigmented (Pgm+) cells of Yersinia pestis is the adsorption of sufficient quantities of exogenous haemin during growth at 26 degrees C to form dark-brown colonies. Carriage of the cloned haemin-storage (hms) locus in pHMS1 restores this phenotype to spontaneous Pgm- chromosomal deletion mutants of Y. pestis. We have mapped the location of the structural genes for four proteins encoded on pHMS1 using minicell, in vitro transcription/translation, and complementation analysis. The hmsH and hmsF genes encode 90 kDa and 72 kDa protein precursors processed to surface-exposed, outer membrane proteins of 86 kDa and 70 kDa, respectively. Beta-galactosidase positive MudII1734 insertions in hmsR suggest that it encodes a protein that is also essential for haemin storage. Finally, the structural gene for a 41 kDa protein lies distal to the hmsH gene but, unlike hmsH, hmsF, and hmsR, its expression is not essential for the Hms+ phenotype in Y. pestis.