To define the ratio of simian immunodeficiency virus (SIV) RNA molecules to infectious virions in plasma, a ramp-up-stage plasma pool was made from the earliest viral RNA (vRNA)-positive plasma samples (collected approximately 7 days after inoculation) from seven macaques, and a set-point-stage plasma pool was made from plasma samples collected 10 to 16 weeks after peak viremia from seven macaques; vRNA levels in these plasma pools were determined, and serial 10-fold dilutions containing 1 to 1,500 vRNA copies/ml were made. Intravenous (i.v.) inoculation of a 1-ml aliquot of diluted ramp-up-stage plasma containing 20 vRNA copies infected 2 of 2 rhesus macaques, while for the set-point-stage plasma, i.v. inoculation with 1,500 vRNA copies was needed to transmit infection. Further, when the heat-inactivated set-point-stage plasma pool was mixed with ramp-up-stage virions, infection of inoculated macaques was blocked. Notably, 2 of 2 animals inoculated with 85 ml of a pre-ramp-up plasma pool containing <3 SIV RNA copies/ml developed SIV infections characterized by high levels of viral replication, demonstrating that "vRNA-negative" plasma collected from macaques in the pre-ramp-up stage is infectious. Furthermore, there is a high ratio of infectious virions to total virions in ramp-up-stage plasma (between 1:1 and 1:10) and a lower ratio in set-point-stage plasma (between 1:75 and 1:750). Heat-inactivated chronic-stage plasma can "neutralize" the highly infectious ramp-up-stage virions. These findings have implications for the understanding of the natural history of SIV and human immunodeficiency virus infection and transmission.