A lipoprotein gene family first identified in Borrelia burgdorferi strain 297, designated 2.9 LP and recently renamed mlp, was found on circular and linear plasmids in the genome sequence of B. burgdorferistrain B31-M1. Sequence analyses of the B31 mlp genes and physically linked variant gene families indicated that mlpgene heterogeneity is unique and unrelated to location or linkage to divergent sequences. Evidence of recombination between B31mlp alleles was also detected. Northern blot analysis of cultured strain B31 indicated that the mlp genes were not expressed at a temperature (23°C) characteristic of that of ticks in the environment. In striking contrast, expression of manymlp genes increased substantially when strain B31 was shifted to 35°C, a temperature change mimicking that occurring in the natural transmission cycle of the spirochete from tick to mammal. Primer extension analysis of the mlp mRNA transcripts suggested that sigma 70-like promoters are involved inmlp expression during temperature shift conditions. Antibodies were made against strain B31 Mlp proteins within the first 4 weeks after experimental mouse infection. Importantly, Lyme disease patients also had serum antibodies reactive with purified recombinant Mlp proteins from strain B31, a result indicating that humans are exposed to Mlp proteins during infection. Taken together, the data indicate that strain B31 mlp genes encode a diverse array of lipoproteins which may participate in early infection processes in the mammalian host.