The use of zebrafish (Danio rerio) as a model organism has seen rapid growth in recent years. Numerous new techniques have emerged for zebrafish, and many protocols used in other model organisms have been adapted to fit the special needs of this species. Zebrafish cell lines, however, have not been studied to a great extent, and their applications remain limited. The PAC2 fibroblast line, isolated from 24-h post-fertilization zebrafish embryos, is one of the few available lines. Here, we provide a basic set of methods for maintenance and handling of PAC2 cells, as well as general procedures for transfection, immunocytochemistry, and protein extraction.