Catalytic properties and potential functional coupling with the adenine nucleotide translocase (ANT) were studied in arginine kinase (AK) from the mitochondria of the heart of the horseshoe crab,Limulus polyphemus. Kinetic constants determined for cytoplasmic AK, AK in mitochondria, and mitochondrial AK tightly bound to membrane fragments were virtually identical, indicating that these enzyme fractions showed minimal differences in terms of catalytic properties. Kinetic constants were also evaluated in intact mitochondria in the presence and absence of oxidative phosphorylation. The K_a and K_ia values for MgATP were identical. These data, coupled with the observation of identical rates of arginine phosphate production at a range of MgATP concentrations under both conditions, indicate that there is no functional coupling of mitochondrial AK with the ANT system. Additional studies showed that mitochondrial AK was more effective than exogenously supplied creatine kinase (CK) in stimulating mitochondrial respiration via phosphagen formation. AK, because of its position within the mitochondrial environment, appears to reduce diffusion problems for ADP by raising the local concentrations in the intermembrane space. Unlike vertebrate CK, where there is good evidence for functional coupling with the ANT system, we find no such functional coupling between AK and ANT inL. polyphemus mitochondria. However, mitochondrial AK probably plays a limited role in regulating respiratory control and ADP homeostasis in this system.