Cell lines derived from zebrafish embryos show great potential as cell culture tools to study the regulation and function of the vertebrate circadian clock. They exhibit directly light-entrainable rhythms of clock gene expression that can be established by simply exposing cultures to light-dark cycles. Mammalian cell lines require treatments with serum or activators of signaling pathways to initiate transient, rapidly dampening clock rhythms. Furthermore, zebrafish cells grow at room temperature, are viable for long periods at confluence, and do not require a CO2-enriched atmosphere, greatly simplifying culture conditions. Here we describe detailed methods for establishing zebrafish cell cultures as well as optimizing transient and stable transfections. These protocols have been successfully used to introduce luciferase reporter constructs into the cells and thereby monitor clock gene expression in vivo. The bioluminescence assay described here lends itself particularly well to high-throughput analysis.