The relative incorporation of thymine and its analogue, 5-bromouracil, into the DNA of a thymine-requiring bacterium was studied under a variety of conditions. Exponentially growing cultures of Escherichia coli strain TAU-bar were transferred to media containing defined amounts of thymine and 5-bromouracil. Following a period of growth the bacteria were harvested and the DNA was isolated. Density distribution analysis in CsCl density gradients was used to determine the relative incorporation of the natural base and its analogue into the newly-synthesized DNA. Under most growth conditions the bacteria exhibited a marked selectivity for thymine over 5-bromouracil. This selectivity was constant for all isolated DNA fragments and it indicated that few regions exist which require thymine to the exclusion of 5-bromouracil. The selectivity for thymine decreased as the incubation temperature was increased: at 51° no selectivity for thymine was observed. The selectivity for thymine did not change with time over the one generation period studied. The relation of these findings to the viability of bacteria which are incorporating 5-bromouracil into their DNA is discussed.