BACKGROUND HTLV antibody screening assays detect both antibodies to the etiological agent of adult T-cell leukemia and tropical spastic paraparesis HTLV-I and to the less pathogenic HTLV-II. It is critical to make a differential diagnosis of the two viruses. OBJECTIVES To design and evaluate synthetic core and envelope-derived peptide enzyme immunoassays (EIA) for serological differential diagnosis. STUDY DESIGN Peptide EIAs were evaluated with a panel of 202 plasma samples comprised of HTLV antibody positive, serologically classified as confirmed, indeterminate, or non confirmed, characterized as HTLV-I, HTLV-II or neither by genomic amplification. The peptide EIA with the best performance was further used to differentiate between HTLV-I and HTLV-II antibodies in 807 samples from 18 countries in four continents and to provide ratios between the two infections. RESULTS The gp46 peptide EIA correctly identified 96.5% of HTLV-I and 98.6% of HTLV-II antibody-confirmed samples. HTLV-I was found exclusively in Japan and Caribbean countries; almost exclusively in Africa. HTLV-II represented 10-25% of samples from Canada, Chile and Venezuela and was predominant in the US. CONCLUSIONS Differential diagnosis between HTLV-I and HTLV-II can be reliably performed using specific peptides from the gp46 envelope protein of each virus.