The proteins of nine strains of Ureaplasrna urealyticum, representing serovars I through IX, were analyzed by using two-dimensional gel electrophoresis and a sensitive silver staining procedure. The electrophoretic patterns of the nine strains tested could be divided into two distinct major groups, one group consisting of serovars 11, IV, V, VII, VIII, and IX (group A) and one group consisting of serovars I, 111, and VI (group B).Ureaplasma urealyticum strains are a serologically heterogeneous group of organisms (16) that are frequently found in human genital tracts. The pathogenic potential of these organisms has been widely disputed, and the significance of their isolation is often unclear (4, 17, 18). The possibility that some strains or biotypes of U. urealyticurn are pathogenic and others are not has been suggested (15), but serological differences have not been clearly associated with pathogenicity (10,15). Deoxyribonucleic acid hybridization (5) and one-dimensional (8, 13) and two-dimensional (9) polyacrylamide gel electrophoretic analyses of [3'S]methionine-labeled proteins have revealed differences among strains of U . urealyticum. In two of these studies (5, 9), the nine recognized serovars could be separated into two major groups. In this report we describe an additional method of analyzing the proteins of U. urealyticurn strains by using two-dimensional electrophoresis and a sensitive silver-based stain which combines with proteins in a fashion that produces reproducible and characteristic colors (12). Our findings confirm previous reports of two groups within the species U. urealyticurn.Eight strains of U . urealyticum originally obtained from F. T. Black, Aarhus, Denmark, and designated serovars I through VIII (3) and one strain obtained from J. A. Robertson, Alberta, Canada, and designated serovar IX (11) were used. These organisms were grown in U-9 broth (14) supplemented with 2% PPLO serum fraction (Difco Laboratories, Detroit, Mich.) at 37°C in an atmosphere containing 5% C02. Cultures in the log phase of growth were harvested by centrifugation at 12,000 x g for 1 h and washed three times with phosphate-buffered saline. The cell pellet obtained from 1 liter of a 20-h culture was sufficient for more than six analyses. Uninoculated medium was incubated and alkalinized to the same degree as growing cultures and was then centrifuged and processed in the same manner. Each strain was grown and tested at least three times.Each washed cell pellet was suspended in six times its volume of sample buffer containing 2% sodium dodecyl sulfate, 2% P-mercaptoethanol, and 1% cyclohexylaminoethanesulfonic acid (Calbiochem-Behring Corp., La Jolla, Calif.) (pH 9.5) and heated at 98°C for 10 min. Electrophoresis in the first dimension (isoelectric focusing) and the second dimension (sodium dodecyl sulfate slab gel electrophoresis) was performed by using the method of Anderson et al. (2), as described by Dunbar et al. (6). Cylindrical isoelectric focusing gels containing ampholines with a wide pH range (pH 3.5 to 10...