In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of <50 copies/ml during highly active antiretroviral therapy (HAART), low levels of plasma virus remain detectable for years by ultrasensitive methods. The relative contributions of ongoing virus replication and virus production from HIV-1 reservoirs to persistent low-level viremia during HAART remain controversial. HIV-1 vaccination of HAART-treated individuals provides a model for examining low-level viremia, as immunizations may facilitate virus replication and sequence evolution. In a phase 1 trial of modified vaccinia virus Ankara/fowlpox virus-based HIV-1 vaccines in 20 HIV-infected young adults receiving HAART, we assessed the prevalence of low-level viremia and sequence evolution, using ultrasensitive viral load (<6.5 copies/ml) and genotyping (five-copy sensitivity) assays. Viral evolution, consisting of new drug resistance mutations and novel amino acid changes within a relevant HLA-restricted allele (e.g., methionine, isoleucine, glutamine, or arginine for leucine at position 205 of RT), was found in 1 and 3 of 20 subjects, respectively. Sequence evolution was significantly correlated with levels of viremia of between 6.5 and <50 copies/ml (P ؍ 0.03) and was more likely to occur within epitopes presented by relevant HLA alleles (P < 0.001). These findings suggest that ongoing virus replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject to CD8؉ T-cell selective pressures.Highly active antiretroviral therapy (HAART) inhibits human immunodeficiency virus type 1 (HIV-1) replication, decreasing plasma viremia below the detection limit (Ͻ50 copies/ ml) of clinically used viral load assays (32). Most patients receiving standard HAART, however, have low levels of viremia (median, 3.1 copies/ml) detectable by more sensitive assays (11,17,22,28,31,34), the source of which remains unclear and controversial. Low-level viremia may arise from viral expression from cellular reservoirs established before HAART (2, 21-23, 25, 29, 31, 34, 46) or from low levels of complete cycles of virus replication (7, 46). Evidence supporting the former includes the stable persistence of low-level viremia during long-term HAART (2,11,23,25,28,31,34), intermingling of HIV-1 sequences from free plasma virus with persistent replication-competent viral sequences in resting memory CD4 ϩ T cells or proviral DNA in peripheral blood mononuclear cells (PBMC) (2,23,34,46), and a lack of decay in low-level viremia with treatment intensification of standard three-drug HAART regimens (10, 24). The contribution of ongoing complete cycles of virus replication during long-term suppressive HAART is supported by findings of sequence evolution with new drugresistant variants in a subset of patients during seemingly effective HAART (7, 46), decreases in plasma viremia with treatment intensification (22), and transient increases in episomal cDNA following treatment intensification with an integrase ...