To determine the cell envelope proteins of Borrelia burgdorferi recognized by immune sera of patients with late Lyme disease, we developed a Borrelia microarray containing proteins encoded by 90 cell envelope genes and their homologs described in the annotated genomic sequence of B. burgdorferi, strain B31. The protein microarray was used to profile the humoral immune response using sera from 13 patients with late Lyme disease and four normal controls. Although there was considerable heterogeneity in the individual sera responses, 25 of the cell envelope proteins were recognized by seven or more samples. Sera from non-infected individuals lacked reactivity against any of the proteins on the array. Among the most antigenic envelope proteins, BLAST search revealed little sequence homology to known microbial proteins from other species. The proteins that were highly seropositive included several members of the Erp gene families, BBA24 (decorin binding protein A (DbpA)) and members of the Borrelia gene family Pfam113 that code for the Mlp lipoprotein gene family. Several novel, uncharacterized B. burgdorferi antigens identified in this study were BBA14, BBG23, BB0108, BB0442 and BBQ03. The accurate diagnosis of Lyme disease depends on correlating objective clinical abnormalities with serological evidence of exposure to B. burgdorferi. A protein array of the envelope proteins of Borrelia burgdorferi may be very useful in specifically identifying patients with Lyme disease. This approach could contribute to a more rapid discovery of antigens not expressed in vitro that may be useful for the development of vaccine and diagnostics.