Elevated serum low density lipoprotein cholesterol (LDL-C) and triglyceride (TG) and decreased high density lipoprotein cholesterol (HDL-C) levels are established risk factors for cardiovascular disease (CVD). To identify quantitative trait loci influencing lipid levels, we conducted genome-wide linkage analyses of total serum cholesterol (TSC), HDL-C, ln-transformed TG (LNTG) and LDL-C levels in 612 individuals from 28 families of the Amish Family Diabetes Study (AFDS). Subjects were genotyped for 373 microsatellite markers covering all 22 autosomes and the X chromosome at an average density of 9.7 centimorgans. All lipid traits exhibited moderate estimated heritability (h2 +/- S.E.): TSC, 0.63 +/- 0.11; HDL-C, 0.54 +/- 0.08; LNTG, 0.37 +/- 0.08; LDL-C, 0.62 +/- 0.10. The highest logarithm of the odds (LOD) score observed was 2.47 (P = 0.0003), at 3p25 for LDL-C. LOD scores exceeding 2.0 (P < 0.001) were also observed at 2p23 (LOD = 2.17) and 19p13 (LOD = 2.23) for LDL-C, and at 11q23 (LOD = 2.03) for LNTG. Three additional regions exhibited LOD scores greater than 1.5, corresponding to a P-value of <0.005. Many of the regions suggestively linked in this genome-wide scan contain genes encoding proteins with established roles in lipid metabolism, including apolipoproteins, peroxisome proliferater-activated receptor-gamma and the LDL receptor.