BACKGROUND: The durability and breadth of human immunodeficiency virus type 1 (HIV-1)-specific immune responses elicited through vaccination are important considerations in the development of an effective HIV-1 vaccine. Responses to HIV-1 envelope subunit protein (Env) immunization in humans are often described as short-lived. METHODS: We enrolled 16 healthy volunteers who had received priming with an HIV-1 subtype B Env vaccine given with MF59 adjuvant 5-17 years previously and 20 healthy unprimed volunteers. Three booster immunizations with a heterologous subtype C trimeric gp140 protein vaccine were administered to the primed group, and the same subtype C gp140 protein vaccination regimen was administered to the unprimed subjects. RESULTS: Binding antibodies and neutralizing antibodies to tier 1 viral isolates were detected in the majority of previously primed subjects. Remarkably, a single dose of protein boosted binding and neutralizing antibody titers in 100% of primed subjects following this prolonged immunologic rest period, and CD4+ T-cell responses were boosted in 75% of primed individuals. CONCLUSIONS: These results demonstrate that HIV-1 protein immunogens can elicit durable memory T- and B-cell responses and that strong tier 1 virus neutralizing responses can be elicited by a single booster dose of protein following a long immunologic rest period. However, we found no evidence that cross-clade boosting led to a significantly broadened neutralizing antibody response.