Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.