The objective of this study was to assess the inhibitory effect of potential negative regulatory elements on human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) activity. This was carried out by pairwise comparisons of reporter gene activities of HIV-LTR-CAT constructs differing in the presence and absence of nef sequences in transient transfection assays. Parallel transfections were performed in two persistently HIV-infected cell lines and the uninfected parental cell lines. The negative regulatory element (NRE) of the LTR did not suppress HIV LTR activity in any of the cell lines examined. However, a non-LTR-derived fragment of the nef gene had a distinct suppressive effect on activity of the full-length LTR in chronically infected astrocytoma cells. A weaker negative effect of this nef partial sequence (nps) was detected in the other cell lines with constructs lacking the NRE. The nps was capable of suppressing LTR activity in trans in chronically infected astrocytoma cells in a concentration-dependent manner. These results stress a negative role of a non-LTR nef partial sequence in a cell-specific manner. In addition, our data indicate that nps functions in trans with promoters unrelated to HIV LTR such as SV40 early and CMV immediate-early promoters.