International regulations prescribe that the absence of avian leucosis viruses (ALV) in avian live virus vaccines has to be demonstrated. Primary chicken embryo fibroblasts (CEF) from special SPF chicken lines are normally used for detection of ALV. The suitability of the DF-1 cell line for ALV-detection, as alternative for primary CEF, was studied in three types of experiments: (1) in titration experiments without cell passage, (2) in experiments with passages in cell cultures according to European Pharmacopoeia requirements, and (3) in experiments with commercial live avian vaccines that had been spiked with known amounts of ALV. In all tests the sensitivity of ALV-A and ALV-J detections on DF-1 cells was at least as high as on primary CEF. The sensitivity of ALV-B detection was always superior when DF-1 cells were used. ALV were detected earlier in all comparative tests when DF-1 cells were used. ALV-A, ALV-B and ALV-J all induced CPE on DF-1 cells, whereas no clear CPE was seen on CEF-cells. For reasons of sensitivity, standardisation as well as reduction of animal use, the data support the use of DF-1 cells to monitor absence of ALV in vaccine virus seed lots or finished products.