Porphyromonas gingivalis W50 was grown in a chemostat at pH 7.3 under haemin-limitation and haemin-excess at a constant mean doubling time of 6.9 h. Outer membranes (OM) were extracted from whole cells using EDTA and compared by SDS-PAGE. Haemin-limited cells expressed novel outer membrane proteins (OMPs) of mol. mass 115, 113 and 19 kDa when samples were solubilized at 100 degrees C. A 46 kDa OMP was observed in haemin-excess cells but not in those from haemin-limited conditions. Tetramethylbenzidine (TMBZ) staining of gels, after OM solubilization at 20 degrees C, was used to detect haemin-binding proteins (HBPs). HBPs were observed only in OM from haemin-limited cells. The major HBP (mol. mass 32.4 kDa) corresponded to a similar sized Kenacid-blue-stained protein which was not observed in haemin-excess-derived OM. Haemin-limited cells and OM displayed a ladder-like series of Kenacid-blue-stained proteins. Lighter TMBZ-stained proteins of mol. mass 51, 53, 56 and 60 kDa, with mobilities corresponding to those of silver-stained LPS components, were observed in haemin-limited OM. No soluble HBPs were detected extracellularly. The greater number of HBPs expressed by cells grown under haemin-limitation may reflect an additional cell surface receptor system for haemin acquisition under low environmental levels of this essential cofactor.