Abstract Carbamoyl phosphate synthetase I (CPSase I) catalyzes the first reaction of the urea cycle in mammalian ureotelic species. The positive allosteric cofactor N-acetyl- l-glutamate (AGA) is required for CPSase I activity and is important for regulation of the urea cycle. A similar enzyme, CPSase III, catalyzes this reaction in fish; CPSase III differs from CPSase I in that it utilizes glutamine as the nitrogen-donating substrate instead of ammonia. AGA also stimulates the CPSase III-catalyzed reaction, but is not absolutely required for activity if the glutamine concentration is high. There has been no report of the presence or function of AGA in fish. Here we report that AGA is present in those species and tissues of fish that have significant levels of CPSase III and urea cycle activity; the levels of AGA were higher in liver of adult gulf toadfish ( Opsanus beta) and spiny dogfish shark ( Squalus acanthias), both of which have high CPSase III activity, than in bass ( Micropterus salmoides) or trout ( Oncorhynchus mykiss), which have much lower or no CPSase III activity, respectively. In the toadfish the levels of AGA in liver and muscle tissue were considerably higher in the fed than in the fasting state, as is observed in mammalian species; in liver, but not in muscle, the level of AGA increased when the toadfish were confined (stressed), which has been shown to induce a ureotelic response. Toadfish muscle had CPSase III and ornithine carbamoyltransferase activities; the increase in AGA concentration in muscle when fed suggests that the presence of these first two enzymes of the urea cycle in muscle may be physiologically significant. The results indicate that the fish investigated have physiologically significant levels of AGA and that the levels correlate with parameters related to urea cycle activity.