Two-dimensional electrophoretic protein profiles of in vivo and in vitro propagated T.pallidum subsps. pallidum Nichols strain were analyzed and compared. This comparative analysis revealed two in vitro synthesized, cytoplasmic cylinder-associated polypeptides with molecular masses 29.5 and 34.7 kDa, pI 5.62, and one in vitro "lost" polypeptide with molecular mass 34.7 kDa, pI 5.34. integral membrane proteins of in vitro and in vivo propagated T. pallidum was identified by phase partitioning with the nonionic Triton X-114, and twelve outer membrane-associated, antigenic proteins were identified in western blots probed with pooled human secondary syphilitic sera. The solubilization of the outer membrane of T. pallidum with Triton X-114 were monitored by electron microscopy. Treatment of freshly harvested 35S labeled T. pallidum with 1% Triton X-114 resulted in solubilization of the outer membrane and reduction of the diameter of the treponemes from .14 +/- .02 micrometers to .095 +/- .003 micrometers. Examination of thin sections of untreated organisms showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated trponemes showed integrity of the cytoplasmic membrane but the loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Integral membrane proteins of Treponema phagedenis were also identified by phase partitioning with Triton X-114, and sizteen cross-reactive, outer membrane-associated, outer membrane-associated, antigenic polypeptides were identified in western blots probed with pooled human secondary syphilitic sera. The results of this study indicate that tow-dimensional protein profiles of in vivo and in vitro propagated T.pallidum are almost identical except for the differences mentioned. This results also indicate that 1% Triton X-114 selectively solubilizes the outer membrane, and the antigenic hydrophobic proteins present in the detergent phrase are located exclusively in the outer membrane.