We studied the constitutive and lipopolysaccharide (LPS)-induced expression of nuclear protein binding to the negative regulatory element (NRE) of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in fresh human monocytes. We demonstrated the existence of a constitutive factor binding to the NRE 73-bp HpaII/HpaII fragment (-216 to -143) whose expression is up-regulated by LPS treatment. Competition experiments with overlapping oligonucleotides covering the HpaII/HpaII fragment and with mutated oligonucleotides mapped the binding within the TTTCATCAC region (-171 to -163). This binding pattern is unique to human monocytes.