When holo-aequorin is injected into zebrafish embryos at the one-cell stage, it is normally depleted by ~24 h post-fertilization (hpf). In order to acquire Ca(2+) signaling information from embryos older than 24 hpf, we have developed a protocol to express apoaequorin transiently in embryos, after which we reconstitute active holo-aequorin in vivo by introducing the cofactor coelenterazine into the developing embryo. This protocol describes the preparation of apoaequorin mRNA, followed by microinjection into embryos and incubation with coelenterazine to reconstitute holo-aequorin.