To determine whether there are structural differences in two topologically separated, biochemically defined mitochondrial populations in rat heart myocytes, the interior of these organelles was examined by high-resolution scanning electron microscopy. On the basis of a count of 159 in situ subsarcolemmal mitochondria (SSM, i.e., those that directly abut the sarcolemma), these organelles possess mainly lamelliform cristae (77%), whereas the cristae in in situ interfibrillar mitochondria (IFM, i.e., those situated between the myofibrils, n = 300) are mainly tubular (55%) or a mixture of tubular and lamelliform (24%). Isolated SSM (n = 374), similar to their in situ counterparts, have predominantly lamelliform cristae (75%). The proportions of crista types in isolated IFM (n = 337) have been altered, with only 20% of these organelles retaining exclusively tubular cristae, whereas 58% are mixed; of the latter, lamelliform cristae predominate. This finding suggests that, in contrast to SSM, the cristae in IFM are structurally plastic, changing during isolation. These observations on >1,000 organelles provide the first quantitative morphological evidence for definitive differences between the two populations of cardiac mitochondria.