The nature of the cell surface components involved in donor cell clumping (Clu+) and the relationship of Clu+ to high frequency conjugal transfer of lactose utilization (Lac) in Lactococcus lactis subsp. lactis ML3 was examined. Lactose positive (Lac+), Clu+ transconjugants, containing a novel 104 kilobase Lac plasmid, were obtained by mating ML3 with LM2301. When used as Lac+ donors in second round matings, these transconjugants transferred Lac at high frequencies ranging from 10-2 to 10-4 transconjugants per donor CFU. Treatment of donor cells with EDTA and EGTA containing solutions or proteolytic enzymes (proteinase K and chymotrypsin A) resulted in a loss of Clu+. By using a direct plate conjugation technique, these treatments also decreased the capacity for transferring Lac at high frequency. Analysis of cell-surface proteins by SOS-PAGE identified a novel protein of approximately 125 kDa which was present in Clu+ transconjugants, but not in non-clumping transconjugants. These results suggest that Clu+ is required for high frequency Lac transfer in ML3 transconjugants, and at least one large protein is involved in Clu+. De novo synthesis requirements of donor cells for conjugal transfer of Lac were tested on direct plate conjugation technique. Results indicate that de novo protein synthesis and RNA synthesis are not required for conjugal transfer of Lac.