The gene encoding the 66 kilodalton (kDa) protein of Borrelia hermsii HS1 was cloned and sequenced. Chromosomal DNA was prepared from purified B. hermsii and used in construction of genomic library. The library was screened for positive clones by 314 bp DIG-labeled probe synthesized on the basis of the part of the sequence of B. hermsii. Positive clone was subcloned into p2ErO vector and was designated as pBH11. pBH11 were subcloned into pBluscript vector and were designated as pBH11-1 (500 bp), pBH11-2 (800 bp), pBH11-3 (600 bp) and pBH11-4 (800 bp). The plasmids were sequenced and determined the nucleotide sequence of p66. The open reading frame of the p66 consisted of 1803 base pairs coding for 600 amino acid protein. The basic information on the p66 gene of B. hermsii HS1 obtained from this study will be useful for further analysis and experiment of pathogenesis of the borrelia.