Mice have a monodisperse high density lipoprotein (HDL) profile, whereas humans have two major subfractions designated HDL(2) and HDL(3). Human apoA-I transgenic mice exhibit a human-like HDL profile, indicating that the amino acid sequence of apoA-I is a determinant of the HDL profile. Comparison of the primary sequence of mouse and human apoA-I and the previously designated "hinge" domain of apoA-I led us to hypothesize that alpha-helices 7 and 8 (7/8) are determinants of HDL subclass distribution. The following proteins were expressed in Escherichia coli: human apoA-I, T7-hAI; mouse apoA-I, T7-mAI; chimeric human apoA-I containing murine helices 7/8 in place of human helices 7/8, T7-hAI(m7/8); and the reciprocal chimera, T7-mAI(h7/8). The recombinant proteins were examined for their association with human plasma HDL subclasses. The results demonstrated that T7-hAI bound HDL(2) and HDL(3) equally well, whereas T7-mAI bound to HDL(2) preferentially. T7-hAI(m7/8) behaved like T7-mAI, and T7-mAI(h7/8) behaved like T7-hAI. Thus, alpha-helices 7/8 are strong contributors to the pattern of HDL subclass association. Self-association, alpha-helicity, cholesterol efflux, and lecithin-cholesterol acyltransferase activity of the recombinant proteins were also assessed. Human apoA-I self-associates more and activates human lecithin-cholesterol acyltransferase better than mouse apoA-I. These differential characteristics of human and mouse apoA-I are not dependent on helices 7/8.