Most unexpectedly, there is now increasing evidence that mitochondria have novel and crucial functions in the regulatory machinery of the growth/differentiation transition, cell-type determination, cellular movement and pattern formation. Here we created rho delta cells with a reduced amount (about 1/4) of mitochondrial DNA (mtDNA) from Dictyostelium discoideum Ax-2 cells, by exposing Ax-2 cells to ca. 30 microg/ml of ethidium bromide (EtBr) in axenic growth medium. Importantly, the rho delta cells exhibited a series of fascinating behaviors: when they were starved, they showed a marked delay of differentiation and stopped their development at the slug stage, thus failing to construct fruiting bodies. Moreover, cell patterning and cell-type proportioning were found to be greatly modified in slugs (referred to as rho delta slugs) derived from rho delta cells. That is, prestalk differentiation was significantly enhanced in rho delta slugs, while prespore differentiation was markedly inhibited. In addition, the clear anterior prestalk/posterior prespore pattern was considerably disturbed in rho delta slugs, presumably because of incomplete sorting between the two types of differentiated cells. After the assay of phototaxis, rho delta slugs also exhibited highly disordered movement towards the light source. Taken together, these results suggest that mtDNA might have important multiple functions in a variety of cellular processes during Dictyostelium development.