About 38% of primary HIV-1 long terminal repeats (LTRs) contain an insertion (consensus: 5prime prime or minute-ACYGCTGA-3prime prime or minute), termed the most frequent naturally occurring length polymorphism (MFNLP). The MFNLP binds several transcription factors and might affect HIV-1 replication and disease progression in infected individuals. However, its relevance for proviral transcription and for HIV-1 replication in primary cells is unclear. We utilized HIV-1 NL4-3 LTR variants to investigate the effect of the MFNLP on 5prime prime or minuteLTR transcriptional activity in various cell types. Notably, viral promoter activity was studied in primary cells in the context of the integrated provirus, using both single cycle assays with pseudotyped Luciferase reporter viruses and replication-competent HIV-1 mutants. Our results demonstrate that the presence, absence, or duplication of the 5prime prime or minute-ACYGCTGA-3prime prime or minute motif has little effect on viral promoter activity in T cell lines, peripheral blood mononuclear cells (PBMC), and monocyte-derived macrophages (MDM). Furthermore, all HIV-1 LTR variants showed efficient induction upon stimulation with TPA and/or ionomycin and replicated with comparable efficiency in a human T cell line and in PBMC. Thus, the MFNLP does not significantly affect HIV-1 5prime prime or minuteLTR transcriptional activity and viral replication in primary cells, suggesting that this common sequence variation has little impact on the clinical course of HIV-1 infection.