Recruiting safe, volunteer blood donors requires understanding motivations for donating and knowledge and attitudes about HIV. We surveyed 1,600 persons presenting for blood donation at a large blood bank in São Paulo, Brazil using a self-administered, structured questionnaire, and classified motivations into three domains as well as categorizing persons by HIV test-seeking behavior. Motivations, in descending order, and their significant associations were: "altruism": female gender, volunteer donor and repeat donor status; "direct appeal": female gender, repeat donor status and age 21-50 years; "self-interest": male gender, age under 20 years, first-time donor status and lower education. HIV test-seekers were more likely to give incorrect answers regarding HIV risk behavior and blood donation and the ability of antibody testing to detect recent HIV infections. Altruism is the main motivator for blood donation in Brazil; other motivators were associated with specific demographic subgroups. HIV test-seeking might be reduced by educational interventions.