Brazil may have the highest absolute number of HTLV-I/II seropositive individuals in the world. Screening potential blood donors for HTLV-I/II is mandatory in Brazil. The public blood center network accounts for about 80.0% of all blood collected. We conducted a cross-sectional study to assess the geographic distribution of HTLV-I/II serological screening prevalence rates in blood donors from 27 large urban areas in the various States of Brazil, from 1995 to 2000. Enzyme immunoassay (EIA) was used to test for HTLV-I/II. The mean prevalence rates ranged from 0.4/1,000 in Florianopolis, capital of Santa Catarina State, in the South, to 10.0/1,000 in São Luiz, Maranhão State, in the Northeast. EIA prevalence rates are lower in the South and higher in the North and Northeast. The reasons for such heterogeneity may be multiple and need further studies.