Derivatives of the lactose miniplasmid pMG820 were constructed in which a staphylococcal erm gene was inserted and in which this was accompanied by subsequent deletion of the lactose genes. The resulting plasmids were thus marked with both erythromycin resistance and lactose utilization genes in pF1132 or solely erythromycin resistance in pF1133. These plasmids retained the normal conjugation properties characteristic of lactose plasmid pLP712, including the generation by intermolecular rearrangement of high-frequency-transfer Clu+ derivatives which exhibited cell aggregation. The use of such Clu+ plasmids in a variety of mating experiments between different lactococcal strains and the observation of cell aggregation when particular mating mixtures were made led to the discovery of a new component of this conjugation system named Agg. A chromosomal gene agg was postulated to be present in some but not all strains of lactococci. High-frequency conjugation and cell aggregation thus depend on the presence of both Agg and Clu, although in a mating pair these components can be in the same or in separate strains. The Agg and Clu components may be analogous to the binding substance and aggregation substance that are involved in the hemolysin plasmid transfer system of Enterococcus faecalis, although control of their expression is different.