In our attempts to transform Lactococcus lactis subsp. lactis with pC194, a staphylococcal chloramphenicol resistance plasmid, only a few transformants could be obtained and only when relatively large amounts of plasmid DNA were used. However, when pC194 DNA from lactococcal transformants was introduced back to Staphylococcus aureus and reisolated, it could be retransformed into L. lactis at substantially higher frequencies. It was concluded that pC194 had undergone mutation expanding its host range. By exchanging DNA fragments between the original pC194 and the variant transforming L. lactis (named pVS41), the mutation could tentatively be located within the 1.1-kbp AccI-HaeIII fragment. Comparison of the DNA sequences in the vicinity of the replication plus-origin revealed the formation of an "opal" stop codon (TGA) apparently interrupting the synthesis of the putative protein C for which no function has been described. Cloning this mutation within a 194 bp AccI-MspI fragment on pC194 made this plasmid able to transform L. lactis. Whether the mutation somehow affects functions controlling plasmid host-specificity, or whether the extended host range reflects, for example, mutational inactivation of some lactococcal restriction site, cannot yet be stated on the basis of these data.