Highly purified mitochondria isolated from 14-day-old pea (Pisum sativum L., cv Little Marvel) seedlings contain a homolog of the 70,000 molecular weight heat-shock protein. The amount of this heat-shock cognate (Hsc70) was not reduced by limited proteolysis of intact mitochondria or by preparation of mitoplasts, indicating that the protein is located within the matrix compartment. Pea mitochondrial Hsc70 binds to immobilized ATP and reacts on western blots with anti-tomato Hsc70 antiserum. When a mitochondrial matrix fraction was incubated with [gamma-(32)P]ATP, there was phosphorylation of Hsc70. The extent of phosphorylation was increased by including calcium chloride in the reactions. Phospho amino acid analysis of purified mitochondrial Hsc70, phosphorylated in the calcium-stimulated reaction, revealed only phosphothreonine. Pea mitochondrial Hsc70, purified by a combination of ATP-agarose affinity chromatography and gel permeation chromatography, was labeled when incubated with ATP plus calcium, suggesting autophosphorylation rather than phosphorylation by an associated kinase. In analogy to mammalian cells and yeast, it is likely that mitochondrial Hsc70 acts as a molecular chaperone, and it is possible that phosphorylation plays a role in chaperone function.