Net phospholipid synthesis is discontinuous during the Caulobacter crescentus cell cycle with synthesis restricted to two discrete periods. The first period of net phospholipid synthesis begins in the swarmer cell shortly after cell division and ends at about the time when DNA replication initiates. The second period of phospholipid synthesis begins at a time when DNA replication is about two-thirds complete and ends at about the same time that DNA replication terminates. Thus, considerable DNA replication, growth, and differentiation (stalk growth) occur in the absence of net phospholipid synthesis. In fact, when net phospholipid synthesis was inhibited by the antibiotic cerulenin through the entire cell cycle, both the initiation and the elongation phases of DNA synthesis occurred normally. An analysis of the kinetics of incorporation of radioactive phosphate into macromolecules showed that the periodicity of phospholipid synthesis could not have been detected by pulse-labeling techniques, and only an analysis of cells prelabeled to equilibrium allowed detection of the periodicity. Equilibrium-labeled cells also allowed determination of the absolute amount of phosphorus-containing macromolecules in newborn swarmer cells. These cells contain about as much DNA as one Escherichia coli chromosome and about four times as much RNA as DNA. The amount of phosphorus in phospholipids is about one-seventh of that in DNA, or about 3% of the total macromolecular phosphorus.