Injection of antisense oligonucleotide morpholinos to elicit selective gene knockdown of ammonia (Rhag, Rhbg and Rhcg1) or urea (UT) transporters was used as a tool to assess the relative importance of each transporter to nitrogen excretion in developing zebrafish, Danio rerio. Knockdown of UT caused urea excretion to decrease by approximately 90%, while each of the Rh protein knockdowns resulted in an approximately 50% reduction in ammonia excretion. Contrary to what has been hypothesized previously for adult fish, each of the Rh proteins appeared to have a similar effect on total ammonia excretion and thus all are required to facilitate normal ammonia excretion in the zebrafish larva. As demonstrated in other teleosts, zebrafish embryos utilized urea to a much greater extent than adults and were effectively ureotelic until hatching. At that point, ammonia excretion rapidly increased and appeared to be triggered by a large increase in the mRNA expression of Rhag, Rhbg and Rhcg1. Unlike the situation in the adult pufferfish (35), the various transporters are not specifically localised to the gills of the developing zebrafish but each protein has a unique expression pattern along the skin, gills and yolk sac. This disparate pattern of expression would appear to preclude interaction between the Rh proteins in zebrafish embryos. However, this may be a developmental feature of the delayed maturation of the gills because as the embryos matured, expression of the transporters in and around the gills increased.