We propose that transcript levels for some genes are affected by the bacterial cell division cycle and this may be an important factor to consider when designing synthetic bacterial genomes. To test this hypothesis, transcript levels of 58 genes in Escherichia coli B/r A were determined at five times during the cell division cycle. A two-step ANOVA technique was used to analyze data from custom oligonucleotide microarrays containing genes involved in important cellular processes including central metabolism, macromolecular synthesis, and transport and secretion. Consistent with results previously found in Caulobacter, approximately 17% of the transcript levels were cell cycle dependent. Cell cycle regulation can be divided into two classes: genes displaying increased transcript concentrations following gene replication and genes displaying an increased transcript concentration prior to replication initiation. Transcripts levels for hns, uspA, and zwf were affected by the cell division cycle, but did not fit well into either class. These results indicate that transcription of a significant fraction of the genome is affected by replication cycle progression. The results also show that both physical gene position and the physiological function of a gene affect when it is transcribed. In addition to the simple association with replication fork progression, other phenomena must be occurring to account for some of our observations. In conclusion, gene position, with regard to the C period, and gene function are important factors to incorporate into design criteria for synthetic bacterial genomes.