Protein P19 encoded by the conjugative resistance plasmid R1, is essential for efficient conjugative DNA transfer and infection by the pilus-specific RNA phage R17. Based on sequence homologies P19 belongs to a family of lysozyme-like virulence factors which are found in type III and type IV secretion systems. In this report we describe the processing and subcellular localization of P19. Pulse-chase experiments were used to demonstrate the processing of P19 by the signal peptidase I of Escherichia coli. Translocation of P19 across the inner membrane was shown by gene 19-phoA fusions. Cell fractionation studies of P19 expressing cells showed the presence of P19 in the membrane compartment. P19 was solubilized with the detergent Sarkosyl indicating an inner membrane localization. Using sucrose density gradient centrifugation to separate inner and outer membranes, P19 was found in both membrane fractions. Taken together, our data suggest that mature P19 is a periplasmic protein which may be attached to the proposed membrane-spanning DNA transport complex.