The high transfer frequency of pheromone-inducible conjugative plasmids of Enterococcusfaecalis in liquid culture is due in part to the formation of mating aggregates. These aggregates result from the interaction of two surface components, aggregation substance (AS), which is plasmid encoded, and the chromosomally encoded binding substance (BS). In the accompanying paper (S.-M. Kao, S. B. Olmsted, A. S. Viksnins, J. C. Gallo, G. M. Dunny, J. Bacteriol. 173:7650-7664, 1991), the sequence of the prgB gene encoding the AS molecule (Asc10) produced by pheromone-induced cells carrying plasmid pCF10 is presented. Here we report the results of genetic and immunological experiments which define the role of Ascl0 in aggregation and plasmid transfer. These data indicate expression of AS on the surface of an E. faecalis cell and its binding to BS expressed on a second cell are required for the formation of a mating pair and the efficient transfer of pCF10 in liquid matings. However, the orientation of the receptors was not critical for transfer; ie., AS expressed on recipient cells could facilitate plasmid transfer via binding to BS on the donor. Our results suggest that additional (as yet unidentified) products are involved in forming the channel that ultimately serves to transfer the DNA, with AS-BS binding serving primarily to generate the initial attachment between cells. The putative prgC gene product, identified by DNA sequencing (data presented in the accompanying paper), could be involved in transfer events occurring subsequent to aggregation.