We investigated effects of site-specific mutation of the putative C/EBP binding site in the feline immunodeficiency virus (FIV) long terminal repeat (LTR) on the basal promoter activity in Crandell feline kidney (CRFK) cells and on replication efficiency in CRFK cells and a T-lymphoblastoid cell line, MYA-1 cells. Mutation of the C/EBP site reduced the basal promoter activity in CRFK cells and prevented efficient FIV replication in both CRFK and MYA-1 cells. Gel-mobility-shift assay using nuclear extracts from CRFK and MYA-1 cells revealed that the nuclear factor(s) actually binds to the C/EBP site, but there was a clear difference in the binding patterns to the C/EBP site between CRFK and MYA-1 cell nuclear proteins. Furthermore, we demonstrated that the C/EBP site is necessary for inhibition of FIV LTR-directed gene expression by pseudorabies virus (PRV) ICP4. The C/EBP site is sufficient to confer inhibitory effect by PRV ICP4 on heterologous promoters. These data suggest that the C/EBP site in the FIV LTR is important for the positive regulation of FIV gene expression and replication and is also required for the negative regulation of FIV gene expression by PRV ICP4.