Whole cell respiration rates were measured polarographically during agitated cultivation of Naegleriafowleri. During log growth, amebae consumed 30 ng atoms O/min/mg cell protein. The ameba's respiration rate gradually decreased 3-fold during stationary phase. Intact mitochondria were isolated from N. fowleri and the oxidative and phosphorylative capacities of the mitochondria were studied with an oxygen electrode apparatus. Mitochondria, stored in isolation medium at 0 C, lost about 20% of respiratory and phosphorylating activity after 4 hr and were most stable at pH 7 to 8. Mitochondria rapidly oxidized glutamate, NADH, pyruvate, succinate, and other Krebs cycle intermediates but slowly oxidized lactate and a-glycerophosphate. The rates of substrate oxidation were ADP dependent and phosphorylative ef- ficiencies (ADP:O ratios) were about 1.4 for NAD-linked substrates and 1.0 for succinate. The respiratory control ratios were 1.5 to 3 for 11 substrates and dependent on the addition of Pi, Mg2+, and serum albumin to the reaction mixture. Cyanide, azide, malonate, and amytal inhibited the oxidative phosphorylation of the present mitochondria essentially the same way as that of the mammalian system while rotenone inhibited both glutamate and succinate oxidation. Pentachlorophenol and DNP uncoupled glutamate and succinate oxidation from phosphorylation. Difference spectra of oxidized and dithionite-reduced mito- chondria showed distinct absorption bands of flavins, c-type, b-type, and a-type cytochromes. Naegleria fowleri is an ameboflagellate which grows and divides as an ameba but may spontaneously transform into a flagellate. Nor- mally free living, it is an opportunistic para- site of humans. Unlike Entamoeba histolyti- ca, an anaerobic parasitic ameba of the large intestine lacking mitochondria (Griffin, 1971), N. fowleri lives in aerobic aqueous environ- ments (DeJonckheere, 1977), has many mito-