Human T-lymphotropic virus type II (HTLV-II) prevalence is very low among the general U.S. population, intermediate among American Indians, and high among injecting drug users and their sexual partners. However, the transmission dynamics underlying this distribution are not well described. We obtained blood specimens from 493 blood donors found to be seropositive for HTLV-II at blood centers in five U.S. cities. Nested polymerase chain reaction was used to amplify a 672-bp region of the HTLV-II long terminal repeat region, and restriction fragment length polymorphism (RFLP) analysis was performed to classify each virus into subtypes as defined by Switzer et al. (1995, J. Virol. 69, 621-632). Associations between RFLP subtype and other characteristics were analyzed using multivariable logistic regression. HTLV-II subtype a0 was independently associated with age over 30 years (odds ratio (OR) = 2.12, 95% confidence interval (CI) 1.13-3.99) and with Black race/ethnicity (OR = 2.00, 95% CI 1.10-3.65 versus Hispanic race/ethnicity). Conversely, HTLV-II RFLP subtypes b4 and b5 were significantly more common among American Indian (OR = 3.77, 95% CI 1.23-11.57) and Other race/ethnicity (OR = 4.22, 95% CI 1.25-14.27, both versus Black race/ethnicity) and at the Oklahoma City blood center (OR = 3.57, 95% CI 1.08-11.84 compared to Washington, DC/Baltimore). There may have been at least two transmission foci of HTLV-II in the United States: a modest HTLV-II subtype a0 epidemic of unknown source in the 1960s and 1970s spread predominantly among Black persons in several geographic areas and a smaller focus of HTLV-II subtypes b4/b5 among non-Black individuals in Oklahoma and perhaps in other areas not examined by this study.