Human plasma contains a multivalent, Kunitz-type proteinase inhibitor termed tissue factor pathway inhibitor (TFPI), which specifically inhibits the action of the factor VII(a) -tissue factor complex in coagulation. In the present study, we examined the distribution and anticoagulant activity of TFPI among plasma lipoprotein subspecies separated by isopycnic density gradient ultracentrifugation; this procedure permitted the simultaneous fractionation of the major lipoprotein classes (very-low-density lipoprotein [VLDL], intermediate-density lipoprotein [IDL], low-density lipoprotein [LDL], high-density lipoprotein [HDL] 2 and 3, and very-high-density lipoprotein [VHDL]). Studies of eight nonnolipidemic subjects revealed two major lipoprotein carriers of TFPI activity: dense LDL subspecies (d=1.039 to 1.063 g/mL) and both dense HDL particles and VHDL (rf=1.133 to 1.190 g/mL), representing 33.8% and 35.9%, respectively, of the total Upoprotein-assodated TFPI activity in plasma. TFPI activity was also associated with lipoprotein(a) (Lp[a]), whose density distribution (</=1.044 to 1.100 g/mL) overlapped that of LDL and HDL]; such association was related to Lp(a)'s particle size and phenotype. VLDL, IDL, and LDL, through LDL, (</= 1.019 to 1.039 g/mL), HDL2(<f= 1.063 to 1.100 g/mL), and light subtractions of HDL, (d=1.100 to 1.167 g/mL) conveyed only 1.8%, 10%, and 18.5%, respectively, of lipoprotein-associated TFPI activity. Such anticoagulant activity was dependent on the presence of TFPI protein. The dense subspecies or HDL, (d=1.133 to 1.167 g/mL) with which TFPI was preferentially associated were small, displayed a cholesteryl ester to protein ratio of -02, and were deficient in phospholipid (13.6% to 18.3%). HDL subspecies of d=1.110 to 1.167 g/mL mainly contained the higher relative molecular mass form of TFPI of 41 kD (a form that is known to be covalently associated with apolipoprotein [apo] A-II) and minor bands of the 35- and 52-kD forms. The second major localization of TFPI was within the hydrated density range of small, dense LDL particles (<f= 1.033 to 1.063 g/mL), which in comparison with light LDL (</= 1.019 to 1.033 g/mL) exhibited a markedly lower proportion of triglyceride and enrichment in cholesteryl ester. Analysb of the ratios of the percent mass of cholesteryl ester to free cholesterol in LDL subtractions showed that the increase in cholesteryl ester content was positively correlated with an increase in TFPI activity (r=.86, /" =5.0013); equally, a positive correlation between an increase in the free cholesterol to protein ratio in LDL and an increase in TFPI activity (r=.89, PS .0006) was detected. In contrast to dense apo A-I-containing lipoprotein particles, dense LDL subspecies of d= 1.039 to 1.058 g/mL mainly transported the 35-kD form of TFPI. We conclude that among the spectrum of apo B-containing lipoprotein subspecies, small, dense LDL particles provide the most favorable surface structure for efficient TFPI binding and equally, for the expression of its anticoagulant activity.