Initiation of replication of the resistance plasmid R1 is carefully regulated by the two negatively acting factors, CopA and CopB. It is shown here that the temperature-dependent runaway-replication phenotype of an R1 plasmid mutant is caused by two point mutations in each of the promoters for the genes of these control factors. Expression of the two genes is affected in the following way: (1) one C-to-T transition in the putative -35 box of the copB-repA operon creates a two- to three-fold stronger promoter from which expression is temperature-dependent; (2) another C-to-T transition in a G + C-rich area immediately downstream from the -10 box of the copA promoter reduces expression of the copA gene three-fold. The phenotypic consequences of the two mutations are discussed in the light of the current model for R1 replication control.