Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-).