To explore the possibility that Streptococcus sanguis aggregation of platelet-rich plasma (PRP) might be mediated by soluble agents, we tested cell-free S. sanguis supernatant for aggregation activity. The supernatant of untreated S. sanguis was without measurable PRP aggregation activity. In contrast, the cell-free supernatant of ATP-incubated S. sanguis produced an immediate wave of PRP aggregation. The supernatant with PRP aggregating activity contained insufficient proteinase to produce a response. The response increased with the time of incubation with ATP. Active supernatant was desalted and chromatographed on nucleotide-calibrated columns of Dowex 1-X8. An active ADP function was identified. The activity was insensitive to dicyclohexylcarbodiimide, but was sensitive to both Ca2+ and Ca2+-Mg2+ chelating agents, cold (4°C), heat (80°C), pH (optimum between pH 7 and 8), apyrase, and sodium molybdate. In addition, preincubation of PRP with adenosine inhibited activity. Strains of viridans streptococci were screened for activity. Aggregation-promoting strains showed two times more activity than did other strains. Although it was not vigorously excluded that the ADP was discharged from the microbes, the existence of an exogenous ATPase on S. sanguis was strongly suggested. The expression of the activity was associated with the lag time to onset of PRP aggregation with intact S. sanguis. This activity could, therefore, be a synergistic promotor of PRP aggregation and an additional virulence factor in endocarditis.