Human immunodeficiency virus (HIV) infection is associated with a significantly increased incidence of pneumococcal pneumonia and concomitant bacteremia. We hypothesized that the predisposition of HIV-infected patients to invasive pneumococcal infection may be related, in part, to an impaired immune response to the pneumococcal antigen pneumolysin (PLY) because PLY facilitates bacterial invasion. We measured serum anti-PLY antibodies in two separate populations of HIV-infected and HIV-seronegative controls, using both an enzyme-linked immunosorbent assay method and a functional assay of antibody inhibition of PLY-induced hemolysis and cytotoxicity. HIV-infected patients in the United States had significantly lower titers of anti-PLY antibodies by both methods than did seronegative control subjects. Moreover, HIV-infected patients in Kenya who later developed pneumococcal bacteremia also had significantly lower anti-PLY antibody levels at baseline compared with seronegative control subjects. We conclude that lower baseline levels of antibodies to PLY are associated with the higher incidence of bacteremic pneumococcal infections among HIV-infected patients.