The pri gene locus of the conjugative broad host range plasmid RP4 maps between coordinates 40.3 and 43.5 and encodes two antigenically related forms of a DNA primase with a molecular mass of 118 and 80 kDa (kilodalton). Genesis of these two products has been examined using Pri+-recombinant plasmids. As shown by deletion analysis, the primase polypeptides are tow separate translation products which arise from an in-phase overlapping gene arrangement. It is suggested that transcription of a set of RP4 genes including the pri gene starts at a promoter site within the Tra1 region. In vivo, RP4 mutant primase can apparently substitute for Escherichia coli primase as demonstrated by measuring suppression of the dnaG3 (ts) mutant.