The mitochondrial compartment of budding yeast (Saccharomyces cerevisiae) is a highly dynamic net-like structure of tubules that constantly undergo fusion and fission. The outer membrane protein Fis1p plays a crucial role in mitochondrial fission. Here we report on the temporal and spatial dynamics of this organelle in wildtype cells and in fis1Delta mutants. Mitochondria of fis1Delta mutants adapt their mitochondrial network to a change in carbon source. We find that the frequencies of apparent matrix separation and fusion events decrease in both wildtype cells and in mutants lacking Fis1Delta upon glucose repression. Matrix separation could be caused by matrix constriction and does not necessarily require fission of the inner or outer membrane. Double-labelling experiments demonstrated that some of these matrix separations in fis1 mutants are due to genuine tubule fissions, whereas others do not involve fission of the outer membrane. The rates of matrix separation in fis1Delta mutants almost approach those of the wildtype, demonstrating that, although apparently involved in outer membrane fission, Fis1p is not crucial for the separation of the mitochondrial matrix. In mutants lacking the GTPase Dnm1p no complete tubule fissions were recorded, although dnm1Delta mutants display matrix separations as well. The data suggest that different molecular machineries are responsible for the separation of the matrix and the fission of the outer membrane in budding yeast.