Description: Although the majority of adult teleosts excrete most of their nitrogenous wastes as ammonia, several fish species are capable of producing urea early in development. In zebrafish, it is unclear whether this results from a functional ornithine-urea cycle (O-UC) and, if so, how it might be regulated. This study examined the spatiotemporal patterns of gene expression of four major O-UC enzymes: carbamoyl phosphate synthase III (CPSIII), ornithine transcarboxylase, arginosuccinate synthetase, and arginosuccinate lyase, using real-time PCR and whole mount in situ hybridization. In addition, we hypothesized that CPSIII gene expression was epigenetically regulated through methylation of its promoter, a widespread mode of differential gene regulation between tissues and life stages in vertebrates. Furthermore, to assess CPSIII functionality, we used morpholinos to silence CPSIII in zebrafish embryos and assessed their nitrogenous waste handling during development, and in response to ammonia injections. Our results suggest that mRNAs of O-UC enzymes are expressed early in zebrafish development and colocalize to the embryonic endoderm. In addition, the methylation status of CPSIII promoter is not consistent with the patterns of expression observed in developing larvae or adult tissues, suggesting other means of transcriptional regulation of this enzyme. Finally, CPSIII morphants exhibited a transient reduction in CPSIII enzyme activity 24 h postfertilization, which was paralleled by reduced urea production during development and in response to an ammonia challenge. Overall, we conclude that the O-UC is functional in zebrafish embryos, providing further evidence that the capacity to produce urea via the O-UC is widespread in developing teleosts.