We have previously defined the promoter of human urokinase-type plasminogen activator receptor (uPAR) gene in a 188-bp fragment between bases -141 and +47 relative to the translation start site. Here, we report that a novel nuclear factor-kappaB (NF-kappaB)-like sequence (5'-GGGAGGAGTC-3') at -45 is located in the uPAR promoter and one of the two DNase I-protected regions, region I between bases -51 and -30. This NF-kappaB-like motif differs at positions 7-9 from the decameric consensus sequences of NF-kappaB (5'-GGGRNNYYCC-3' where R indicates A or G, Y indicates C or T, and N indicates any nucleotide) and at positions 1 and 7-9 from the kappaB-like motifs (5'-HGGARNYYCC-3' where H indicates A, C, or T, R indicates A or G, Y indicates C or T, and N indicates any nucleotide). Nuclear extracts from HCT116 cells contain proteins that specifically bind to the NF-kappaB-like site at position -45. Mutation of the NF-kappaB-like motif decreased the binding of transcription factor NF-kappaB and reduced the uPAR promoter activity in comparison with the wild-type sequences. Co-transfection with a dominant negative I-kappaB kinase-2 expression vector reduced uPAR promoter activity by 65-75%. These results demonstrate that a previously uncharacterized NF-kappaB motif is required for uPAR promoter activity.