Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. In this paper the potential coupling of mitochondrial metabolism to mitochondrial protein synthesis was investigated and the concentration of nucleotides and substrates for optimal translation in isolated rat heart mitochondria was determined. It was demonstrated that optimal isolated heart mitochondrial protein synthesis required the presence of an oxidizable substrate. Of the substrates tested, glutamate (20 mM) supported translation best followed by malate, succinate, and alpha-ketoglutarate, whereas pyruvate supported synthesis poorly. Unlike other recent mammalian mitochondrial systems, the presence of an oxidizable substrate was required for translation even in the presence of medium ATP and an exogenous energy-generating system. Mitochondrial translation also required the presence of adenine nucleotide that could be added as ADP or ATP; however, ATP added above 0.5 mM became progressively inhibitory. As a result, synthesis was supported significantly better by ATP synthesized by the system from added ADP, than by ATP added directly to the system. However, if the phosphorylation of ADP was prevented by limiting the phosphate concentration, ADP itself strongly inhibited mitochondrial protein synthesis. This inhibition appeared to be closely related to the energy charge of the system rather than to absolute levels of ADP, indicating for the first time that mitochondrial translation, like its cytoplasmic counterpart is regulated by energy charge. Last, this system did not require the inhibition of guanine nucleotide or exogenous energy-generating systems.(ABSTRACT TRUNCATED AT 250 WORDS)