Several envelope proteins of Bordetella pertussis demonstrated differences in electrophoretic mobility, depending upon solubilization temperature before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins were exposed on the cell surface as judged by their accessibility to radiolabeling with 125I. Monoclonal antibodies to two of the heat-modifiable proteins (M(r)s of 18,000 and 91,000) reacted with intact cells in immunofluorescence microscopy experiments, also indicating surface exposure of these two proteins. Two-dimensional gel electrophoresis revealed that two heat-modifiable proteins (a major protein with an M(r) of 38,000 and one with an M(r) of 18,000) migrated as higher-M(r) moieties when solubilized at low temperatures (25°C). Three proteins (M(r)s of 91,000, 32,000, and 30,000) and possibly a fourth (31,000) migrated as lower-M(r) species when solubilized at 25°C, as revealed in the two-dimensional gel system; these three proteins were found only in virulent B. pertussis and were not detected in a phase IV avirulent strain nor in a strain modulated to phenotypic avirulence by growth in nicotinic acid. The 38,000 molecular-weight protein (38K protein) and a 25K protein were found to be noncovalently associated with the underlying peptidoglycan. Small amounts of the 91K and 18K proteins were also found associated with peptidoglycan.