Influences of the cell system on observed EC(50) values of different agents against feline immunodeficiency virus (FIV) were assessed. The activity of various nucleoside reverse transcriptase inhibitors (NRTI) against a lymphotropic FIV strain was evaluated using monocultured thymocytes and a DC-thymocyte coculture. In the second set of experiments activity of carbohydrate binding agents (CBA) towards FIV strains derived from different cell lines (e.g. Crandall feline kidney cells (CRFK) and thymocytes) was compared. We examined three different FIV-based antiviral evaluation systems and obtained marked differences in EC(50) values, especially for CBA entry inhibitors. Our study confirms and extends earlier observed differences between cell systems used for the evaluation of the activity of antivirals towards FIV.