Phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide-binding protein gene family. Expression of PLTP has been implicated in the development of atherosclerosis. We evaluated the effects of PLTP region tagging single nucleotide polymorphisms (SNPs) on the prediction of both carotid artery disease (CAAD) and PLTP activity. CAAD effects were evaluated in 442 Caucasian male subjects with severe CAAD and 497 vascular disease-free controls. SNP prediction of PLTP transfer activity was evaluated in both a subsample of 87 subjects enriched for an allele of interest and in a confirmation sample of 210 Caucasian males and females. Hemoglobin A1c or insulin level predicted 11-14% of age- and sex-adjusted PLTP activity. PLTP SNPs that predicted approximately 11-30% of adjusted PLTP activity variance were identified in the two cohorts. For rs6065904, the allele that was associated with CAAD was also associated with elevated PLTP activity in both cohorts. SNPs associated with PLTP activity also predicted variation in LDL-cholesterol and LDL-B level only in the replication cohort. These results demonstrate that PLTP activity is strongly influenced by PLTP region polymorphisms and metabolic factors.