We investigated possible biochemical pathways explaining extreme ammonia tolerance by the gulf toadfish and specifically tested the prediction that the gulf toadfish Opsanus beta is more tolerant than the plainfin midshipman Porichthys notatus, two confamilial species, because it reverses brain glutamine accumulation during high ammonia exposure. This prediction stems from previous studies demonstrating that gulf toadfish produce urea from glutamine, a pathway not present in the ammoniotelic midshipman. Our results show that at the same water NH(3) concentration, ammonia increases more from control levels in brains of midshipman than toadfish. After 48 h of exposure to 50% of their respective LC(50) (96 h) value for ammonia, toadfish are able to return the ammonia-induced increase in brain glutamine back to control values, reducing brain glutamine by 2,500 micromol kg(-1). However, in midshipman, brain glutamine remains significantly elevated from control throughout the experiment. Toadfish exposed to 33% of their LC(50) (96 h) showed an initial increase in whole body urea, which is then reduced at a rate of 104 micromol kg fish(-1) h(-1) and could be directly excreted into the water. We discuss how the special handling of glutamine in toadfish may explain in part their great tolerance to ammonia.