Blood samples were obtained from 16 hemophiliacs who had a 50%-94% defined risk of human immunodeficiency virus (HIV) type 1 infection on the basis of treatment history and from 14 controls not at risk for HIV infection. HIV-1 was not detected in any of 12 patient samples by cocultivation nor in 14 patient samples by the polymerase chain reaction. Peripheral blood cells from 7 seronegative hemophiliacs at highest risk of seroconversion (94%) were less susceptible to HIV-1 infection in vitro than were cells from healthy controls (P <.025, one-tailed Wilcoxon rank sum test). In contrast, the susceptibility to HIV-1 infection of lymphocytes from 6 seronegative hemophiliacs at moderate risk (50%-56%) of seroconversion did not differ from that of cells from controls or from high-risk hemophiliacs. Therefore, prolonged periods of seronegative HIV-1 infection are not common in this high-risk population. In addition, among hemophiliacs there may exist heterogeneity in susceptibility to HIV-1 infection in vitro and in vivo.