The positions of the 64 DNA tri-nucleotides (triplets) along the Borrelia burgdorferi chromosome were determined and cumulative position plots (CPP) were obtained. Analysis of CPP for complementary triplets revealed close correlations in complementary triplet frequencies (CTF) between opposing leading and lagging strands. Such bilateral inverse symmetry (BIS) applied also to complementary mono- and di-nucleotides and to some >3 n-tuples. At the level of individual bases BIS explains Chargaff's second parity rule for whole bacterial chromosomes. Using shuffled control sequences we show that single-base BIS was not the source of higher-order BIS. Analysis of CTF in 45 other chromosomes suggests that BIS is a general property of eubacteria. BIS at the various levels may be due to the very similar numbers of codons used in chromosomal halves. Evolutionarily, BIS could have resulted from asymmetric substitution of bases combined with genetic rearrangements. However, the provocative theoretical alternative of whole-genome inverse duplication is here considered. (C) 2002 Elsevier Science (USA). All rights reserved.