The Escherichia coli DNA replication genes dnaZ and dnaX have previously been localized very near each other at 10.4 to 10.5 min on the chromosome map. These genes were cloned from a dnaZ+X+ plasmid of the Clarke and Carbon collection by identifying complementing fragments and both were located on a 2.1 kilobase pair (kb) fragment. The organization of the Z and X genes was investigated by Tn5 mutagenesis of a Z+X+ plasmid. Insertions which abolished Z or X complementing activity were mapped by restriction enzyme analysis within the 2.1 kb fragment. With the exception of one atypical insertion, all the insertions inactivated both Z and X complementation. The protein products of the dnaZ-dnaX region were labelled in minicells containing dnaZ+X+ and dnaZX::Tn5 plasmids. The 2.1 kb ZX region (which has a maximum coding capacity of 77,000 daltons of protein in a single reading frame) directed the synthesis of two proteins, one of 75,000 daltons, designated dnaX, and another of 56,500 daltons, designated dnaZ. Tn5 insertion into the ZX region interrupted the synthesis of these proteins; the detection of truncated fragments of dnaX determined the direction of transcription. In vitro, using a coupled transcription-translation system dependent on plasmid DNA, synthesis of the 75,000 dalton dnaX protein was demonstrated, but there was no detectable synthesis of the smaller dnaZ protein. Probably, therefore, the 75,000 dalton dnaX protein is cleaved in vivo to generate the dnaZ protein. It is possible that the 75,000 dalton product is the tau subunit of DNA polymerase III because they migrated similarly in electrophoresis.