The activation state of CD4(+) T cells plays a crucial role in the establishment of a productive human immunodeficiency virus infection. Here, we show that T cells stimulated for 1 day demonstrated delayed kinetics of viral reverse transcription and integration compared to cells stimulated for 2 days prior to infection. As a result, the efficiency of reverse transcription and integration inhibitors differs in these differentially stimulated cells. These studies increase our understanding of how T cells support viral replication and provide insight regarding the efficiency of antiretroviral therapy in lymphoid compartments.