The protein composition of the virulent M129 strain of Mycoplasma pneumoniae was compared to that of its homologous avirulent strain by the use of standard one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Forty-nine individual M. pneumoniae cell proteins were resolved by this method, and the virulent strain was shown to possess a single high-molecular-weight protein not present in avirulent cells. Variability in the resolution of this particular protein in one-dimensional gels prompted the application of two-dimensional gel electrophoresis to the analysis of M. pneumoniae cell proteins. The sequential use of isoelectric focusing in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension permitted the resolution of a least 142 individual M. pneumoniae cell proteins. Application of nonequilibrium pH gradient electrophoresis in the first dimension achieved the resolution of at least 20 additional basic proteins. Three proteins which are synthesized only by cells of the virulent strain, and not by the homologous avirulent strain, were identified by these two-dimensional gel electrophoresis techniques.