A polA mutant of Mycobacterium smegmatis (Ms) lacking DNA polymerase activity could not support the replication of a pAL5000-derived plasmid or a derivative harbouring the RLEP-flanked polA gene from M. leprae (Mlep). In contrast, the plasmid containing the M. tuberculosis polA gene transformed the mutant with high efficiency and complemented its damage-sensitive phenotype suggesting that the replication of the pAL5000 origin is dependent on host PolI function and that the RLEP-flanked Mlep polA gene is not expressed in Ms.