Any enzyme or enzyme system that produces ADP in proximity to the mitochondria may be capable of stimulating respiration. Hexokinase (HK), adenylate kinase (AK), and mitochondrial creatine kinase (Mi-CK) all catalyze reactions that produce ADP and thus may play a role in cellular nucleotide metabolism or control of mitochondrial oxidative phosphorylation. Respiratory characteristics and enzyme activities of mitochondria simultaneously isolated from heart and uterus of the gravid guinea pig were compared. The abilities of AMP, glucose, and creatine to stimulate mitochondrial respiration via AK, HK, and Mi-CK systems, respectively, were examined. Although the uterine Mi-CK activity is low compared with the values found in heart, the activities of HK and AK were significantly greater. Furthermore, the abilities of HK and AK to stimulate respiration (functional activity) were greater in the uterine mitochondria. Indeed, the activity of AK was sufficient to generate maximal (state 3) respiration. The apparent Michaelis constant (Km) for ADP to stimulate respiration in the isolated uterine mitochondria was significantly different from that of the heart mitochondria (9.6 +/- 0.9 and 5.1 +/- 1 microM ADP, respectively). It is concluded that uterine mitochondria can use HK and AK systems in addition to the CK system in enhancing local ADP concentration, which may aid in the mitochondrial responses to energetic demands.