Borrelia hermsii undergoes spontaneous antigenic variation in vivo and in vitro. Serotype specificity is associated with expression of one of a family of molecular weight-variable proteins, the pI proteins. We studied the structure of the pI proteins as well as the molecular weight-invariable pII proteins of three serotypes of B. hermsii HS1: C, 7, and 21. The techniques used were one-dimensional (1-D) mapping of Staphylococcus aureus V8 protease-generated peptides and two-dimensional (2-D) mapping of alpha-chymotrypsin-generated peptides. The pI and pII proteins were isolated by excision of polypeptides from stained polyacrylamide gel electropherograms. The 1-D peptide patterns were visualized by fluorography of intrinsically [14C]leucine-labeled proteins or by silver stain. Before 2-D mapping, polypeptides in excised gel fragments were labeled with 125I in the presence of chloramine-T. We also compared the 2-D peptide maps of pI proteins, pI7 and pI21, after their surface-exposed portions were radioiodinated using 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril (Iodogen). The I-D and 2-D peptide maps demonstrated the following: (a) pI proteins of the three serotypes have few V8 protease- or chymotrypsin-generated peptides in common, and (b) pI proteins of each serotype appear to be identical. The findings suggest that pI protein variability derives from extensive differences in the amino acid sequences of these proteins.