Annexin was purified from rat liver mitochondria to an apparent homogeneity with a molecular weight of 35kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified mitochondrial annexin (AXmito) was identified as annexin I by an immunoblot analysis using anti-annexin I antibody. The inhibitory effect of AXmito I on porcine pancreatic phospholipase A, activity was as potent as that of bovine lung annexin I. The presence of annexin in mitochondria was confirmed by an electronmicroscopic study. AXmito I was shown to be phosphorylated by intrinsic protein tyrosine kinases on its tyrosine residues. This annexin was also phosphorylated by protein kinase C.