Cats harbor an infectious endogenous retrovirus, named RD114 virus. It is therefore necessary to monitor RD114 virus production in feline cells which are used for biological products as substrates. In this study, a feline sarcoma-positive leukemia-negative (S+L-) fibroblast cell line, named QN10S cells, was found to be highly susceptible to RD114 pseudotype viruses. The cells were transformed by infection with RD114 virus and the numbers of foci could be counted. The sensitivity of the focus assay was lower than that of the LacZ marker rescue assay in detecting RD114 virus. Although the assay is not suitable to detect a small amount of the virus, the assay will be useful for virological studies of RD114 virus.