Although many researchers have worked with oxygen alone or substrate alone, there has been one attempt to relate an animal’s respiratory physiology to substrate and this, in turn, to distribution. The present study was undertaken to investigate further this relationship and some of its implications. The burrowing nymphs of the mayflies Hexagenia limbata and Ephemera simulans were selected for study because they exhibit in their distribution the experimentally useful characteristic of inhabiting different yet intergrading substrates. Objectives of this investigation were to determine for each species (1) the preferred substrate as measured by laboratory and field observations and experiments, (2) the effects of oxygen concentration and particle size of substrate on oxygen consumption, and (3) the effects of morphological adaptations and behavioral characteristics on substrate selection and respiratory control. A function of these objectives would be to elucidate the respiratory similarities and differences of the two species. Such traits certainly function in combination with morphology and behavior and the substrate and oxygen parameters. Hence, all of these factors can be compared and related in an attempt to show their role in the distribution of the two mayfly species.