In vitro replication of the golden shiner virus (GSV) occurred at 20 C to 30 C in fathead minnow cells; GSV did not replicate at 15 C or 35 C. Optimal replication temperature was approximately 30 C, where the latent period was 8 hr and viral yield was 113 infectious units per cell. Maximum cytopathic effect coincided with maximum released viral titer. Suitable storage temperatures for GSV were −70 C and 4 C. Viral infectivity was rapidly lost at temperatures above 20 C and at normal freezer temperature (−15 C).