The neuronal ceroid-lipofuscinose (NCL) are recessively inherited lysosomal storage diseases in children and animals. The major stored protein in many of these diseases is subunit c of the mitochondrial inner membrane H+-transporting ATP-synthase. Previous studies of naturally occurring ovine ceroid-lipofuscinosis (OCL) in South Hampshire sheep showed that the genes and transcripts for subunit c were normal and inferred that this protein was expressed normally in mitochondria prior to storage in lysosomes. Accumulation in mitochondria has not been conclusively established and we have therefore used the South Hampshire model to demonstrate approximately 1.8-fold normal levels of subunit c in mitochondrial inner membranes prepared from liver. Other mitochondrial inner membrane and ATP-synthase proteins that could be detected by mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) were present in normal amounts. The accumulating subunit c showed normal post-translational modification but was abnormally resistant to proteolysis. These results are consistent with the hypothesis that OCL may result from a mitochondrial disorder that affects turnover of correctly expressed subunit c, although we cannot exclude the possibility that a postmitochondrial defect delays processing of subunit c out of mitochondria.