The effect of chromosomal location on the expression of bacterial genes is understudied; here, Bryant et al. re-address how chromosomal position affects gene expression in Escherichia coli K-12. By inserting a reporter cassette composed of GFP under the control of the lac promoter at 14 different positions in the genome, they show that gene expression varies by ∼300-fold depending on the insertion site; the expression of some loci was silenced or was increased compared with insertion at the wild-type lac locus. The differences in gene expression levels were not solely due to gene copy number and instead were mediated at the transcriptional level. In addition, DNA gyrase activity was associated with loci in which gfp was highly expressed, whereas transcriptionally silenced regions were associated with the loss of gfp expression. Thus, transcription as well as local chromatin structure contribute to the chromosomal position effect on gene expression.