The rate of generation of Escherichia coli B/r is inhibited by 5-fluorouracil (FU) and this rate is halved by 30μg/ml at 37.5°C in peptone broth USP. The FU is highly bound to the nutrients of this medium. The activity of FU is 1,000 times greater in non-binding casamino acids medium. 5-Fluorodeoxyuridine is less active. The drug-affected steady state generation in peptone broth is achieved 40 min (2 generations) after drug addition and conforms to N = No ekappt where N is the number of organisms per unit volume at time t, and kapp is the new generation rate constant. The new kapp was achieved after 20 min in the casamino acid medium but started to increase in magnitude after 100 min. These kapp values are non-linear functions of drug concentrations and approach zero at high concentrations consistent with kapp = ko-ka[FU]/(1+kb[FU]). The total (Coulter) and viable (Colony) counts were coincident for all doses studied except for the case of complete inhibition in casamino acid medium so that the usual action of FU on E. coli is inhibition of microbial generation without any apparent death of drug-affected organisms. A complete pH range study shows that the non-protonated species is the active form of the compound. The effect of FU is competitively inhibited by uracil, uridine and deoxyuridine, that are inactive by themselves. Thymidine potentiates the inhibitory effect of FU in peptone broth only but is ineffective by itself. 5-Chloro-, 5-bromo-, and 5-iodouracil have no inhibitory effect and the former two do not antagonize the action of FU. There is no significant effect of inoculum size below 106 organisms/ml on the generation inhibition of FU. There appears to be a significant effect of nutrient concentration in peptone broth on FU-affected generation rate, possibly due to drug-binding of nutrients or the presence of FU antagonists in the media.