The objectives of this study were (1) to determine the type of carbamoyl phosphate synthetase (CPS) present, and the compartmentalization of arginase, in the livers of the African lungfishes, Protopterus aethiopicus and Protopterus annectens, and (2) to elucidate if these two lungfishes were capable of increasing the rates of urea synthesis and capacities of the ornithine-urea cycle (OUC) during 6 days of aerial exposure without undergoing aestivation. Like another African lungfish, Protopterus dolloi, reported elsewhere, the CPS activities from the livers of P. aethiopicus and P. annectens had properties similar to that of the marine ray (Taeniura lymma), but dissimilar to that of the mouse (Mus musculus). Hence, they possessed CPS III, and not CPS I as reported previously. CPS III was present exclusively in the liver mitochondria of both lungfishes, but the majority of the arginase activities were present in the cytosolic fractions of their livers. Glutamine synthetase (GS) activity was also detected in the hepatic mitochondria of both specimens. Therefore, our results suggest that the evolution of CPS III to CPS I might not have occurred before the evolution of extant lungfishes as suggested previously, prompting an examination of the current view on the evolution of CPS and OUC in vertebrates. Aerial exposure led to significant decreases in rates of ammonia excretion in P. aethiopicus and P. annectens, but there were no accumulations of ammonia in their tissues. However, urea contents in their tissues increased significantly after 6 days of aerial exposure. The estimated rates of urea synthesis in P. aethiopicus and P. annectens increased 1.2- and 1.47-fold, respectively, which were smaller than that in P. dolloi (8.6-fold) reported elsewhere. In addition, unlike P. dolloi, 6 days of aerial exposure had no significant effects on the hepatic CPS III activities of P. aethiopicus and P. annectens. In contrast, aerial exposure induced relatively greater degrees of reductions in ammonia production in P. aethiopicus (34%) and P. annectens (37%) than P. dolloi (28%) as previously reported. Thus, our results suggest that various species of African lungfishes respond to aerial exposure differently with respect to nitrogen metabolism and excretion, and it can be concluded that P. aethiopicus and P. annectens depended more on reductions in ammonia production than on increases in urea synthesis to ameliorate ammonia toxicity when exposed to terrestrial conditions. J. Exp. Zool. 303A:354–365, 2005. © 2005 Wiley-Liss, Inc.