Isolation and characterization of the platelet aggregation inhibitory factor of bromelain have been presented in this study. Commercial bromelain consists of 3 major components as demonstrated by discontinuous sodium chloride gradient chromatography through carbixymethyl-sephadex column. Fraction I constituted approximately 19% of the total fraction. This fraction had no proteolytic activity or platelet aggregation inhibiting activity, but showed peroxidatic activity. Fraction II and III, which constituted the remainder of the fraction eluted with 135 mM and 800 mM NaCl concentrations, respectively, showed both proteolytic and inhibition of platelet aggregation, but no peroxidatic activity. Immunoelectrophoresis and polyacrylamide electrophoresis showed fraction I with beta-mobility while fraction II and III demonstrated gamma-mobility. It is suggested that the proteolytic activity is associated with the inhibition of platelet aggregation, since oxidation of fractions II and III with sodium tetrathionate abolished both activities. The mechanism of inhibition of platelet aggregation by bromelain is presently unknown but may involve its influence on the prostaglandin synthetic pathway of platelets.