A new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis subsp. lactis biovar diacetylactis strain DB0410. This plasmid is responsible for the sensitivity of DB0410 to the toxic pyrimidine analogue 5-fluoroorotate. The complete nucleotide sequence has been determined and amounts to 16 404 bp. Of 15 ORFs encountered, three were found to be insertion sequence (IS) elements, identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their C-terminal region. Homologues for four ORFs were found in the IL1403 sequence: the copB gene, coding for a copper-potassium-transporting ATPase B, and the ysbA, ysbB and ysbC genes. The structural organization of the pDBORO replication region is highly similar to other theta-replicating plasmids in both the cis- (repA) and trans-acting (repB and orfX) sequences. By plasmid deletion analysis and molecular cloning, a single locus on pDBORO was found to confer sensitivity to 5-fluoroorotate. It was identified as ysbC, but renamed oroP in order to reflect its function. The oroP gene was found to be essential for the utilization of orotate as the sole pyrimidine source in a strain deficient in pyrimidine de novo synthesis. The amino acid sequence encoded by the ORF showed the characteristic features of a membrane protein. Therefore, oroP most probably encodes an orotate transporter. Surprisingly, homologues of oroP could be identified in the genomes of both L. lactis MG1363 and L. lactis IL1403 despite the fact that these strains were unable to significantly utilize orotate. Cloning of oroP in Escherichia coli and Bacillus subtilis showed that the orotate transport phenotype could be transformed to both organisms. The findings presented indicate that oroP can be used as a powerful, food-grade selection/counterselection marker in many different organisms.