Sequencing of the complete Bacillus subtilis chromosome revealed the presence of approximately 4100 genes, 1000 of which were previously identified and mapped by classical genetic crosses. Comparison of these experimentally determined positions to those derived from the nucleotide sequence showed discrepancies reaching up to 24 degrees (approximately 280 kb). The size of these discrepancies as a function of their position along the chromosome is not random but, apparently, reveals some periodicity. Our analyses demonstrate that the discrepancies can be accounted for by inaccurate positioning of the early reference markers with respect to which all subsequently identified loci were mapped by transduction and transformation. We conclude (i) that specific DNA sequences, such as recombination hotspots or presence of heterologous DNA, had no detectable effect on the results obtained by classical mapping, and (ii) that PBS1 transduction appears to be an accurate and unbiased mapping method in B. subtilis.