The first events of lambda plasmid replication in vivo, which probably regulate this process, are the transcriptional activation of the origin of replication by RNA polymerase and the binding of the initiator protein, lambda O, to this nucleotide sequence. The lambda O protein is known for its rapid proteolytic degradation; hence amino acid starvation of Escherichia coli should result in inhibition of lambda plasmid replication caused by inhibition of protein synthesis. However, contrary to this prediction, we found that lambda plasmid replication, as measured by the increase in plasmid content per bacterial mass, proceeds for hours in an amino acid-starved, relaxed mutant, whereas it is inhibited in its wild-type stringent partner. lambda plasmid replication in amino acid-starved, relaxed cells reveals absolute lambda O dependence and is not inhibited by chloramphenicol at 200 micrograms/ml. This process also occurs in wild-type cells treated with chloramphenicol. We conclude that lambda plasmid replication is under stringent control, probably as a result of the action of ppGpp, the indirect product of the relA gene, on RNA polymerase. The problem of stability of the lambda O initiator protein is discussed.