By photoaffinity labeling human low density lipoproteins (LDL) with [125I]T4we confirmed our previous observation that of the three T4 binding sites of apolipoprotein B-100 (apoB-100) one is in its 26% NH2-terminal portion [apoB- 26, the 140 kDa fragment, residues 1-1297] and two in the remaining 74% COOH portion (apoB-74, 410 kDa, residues 1298-4536). We now show that of these two sites one is in the NH2portion of apoB-74 (apoB-44, 240 kDa, residues 1298-3249) and the other in the nonoverlapping COOH portion (apoB-30, 170 kDa, residues 3250-4536).ApoB-100 contains 13 binding sites for heparin, a known inhibitor of T4binding to the major T4carrier plasma proteins; however, heparin failed to inhibit T4binding to apoB-100 and fragments thereof. The same failure was seen with three monoclonal antibodies (MAbs), 4G3, 5E11, and 43 that block totally or partially LDL binding to the LDL receptor [respective epitopes at residues 2980-3084 (apoB-44), 3441-3569, and 4027- 4081 (apoB-30)]. Of the other 3 MAbs, all without effect on LDL binding to the LDL receptor, [1D1, 2D8, and 16, respective epitopes at residues 474-539 (apoB-26), 1438-1481 (apoB-44), and 4154-4189 (apoB-30)], only two (MAbs 1D1 and 2D8) inhibited T4binding (21 to 39%).We conclude that the three T4-binding sites of apoB-100 are outside the LDL receptor binding domain, distant from the heparin binding sites and, assuming no allosteric effects, in the vicinity of residues 474-539 (T4site of apoB-26), 1438-1481 (T4site of apoB-44), and in the C terminal quarter of apoB-30. © 1990 by The Endocrine Society.