Cells of the monocyte-macrophage lineage play an important role in human immunodeficiency virus type 1 (HIV-1)-associated disease. Infected myeloid precursor cells of the bone marrow are thought to be a viral reservoir that may repopulate the peripheral blood, central nervous system (CNS), and other organ systems throughout the course of disease. To model select aspects of HIV-1 infection of the bone marrow compartment in vitro, the erythro-myeloid precursor cell line, TF-1, was used. Phorbol 12-myristate 13-acetate (PMA) was found to induce the TF-1 cell line to differentiate through the myeloid lineage and become activated, as demonstrated by cellular morphologic changes and surface expression of differentiation and activation markers. Herein we demonstrate that HIV-1 long terminal repeats (LTRs) from T-, M-, and dual-tropic molecular clones have similar basal LTR activity in TF-1 cells and that differentiation of these cells by PMA resulted in increased LTR activity. Examination of specific cis-acting elements involved in basal and PMA-induced LTR activity demonstrated that the transcription factor families nuclear factor-kappa B (NF-kappaB) and specificity protein (Sp) contributed to the LTR activity of TF-1 cells, the Sp family being the most critical. These studies elucidate the impact of infected bone marrow monocytic cell differentiation on LTR activity and its potential impact on HIV-1-associated disease.