Mechanisms for coexistence among micro-organisms were studied by using a species-defined microcosm, consisting of the bacterium Escherichia coli, the ciliate Tetrahymena thermophila and the alga Euglena gracilis. These organisms were chosen as representative of ecological functional groups i.e. decomposer, consumer and producer, respectively. Direct and indirect interactions among these organisms were evaluated by comparisons of their population dynamics in culture with different combinations of the three species. There was an E. coli cell density dependent predator–prey interaction between T. thermophila and E. coli which was only established when there were more than 106 cells ml−1 of E. coli. Indirect interactions were evaluated from the cultivation of each organism in media containing metabolites of the others. Metabolites from each population strongly accelerated the growth of their own populations and those of the others except for the self-toxicity effect of E. coli metabolites. These observations suggested that not only the cell–cell contact of direct interactions, but also metabolite-mediated indirect interactions supported the maintenance of the populations of each micro-organism and their coexistence. In natural ecosystems, there are many interactions and it is difficult to evaluate all those regulating community dynamics. The gnotobiotic microcosm used in this study was shown to be suitable for examining the specific, species–species microbial interactions.