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We evaluated overlap in microhabitat use between nonnative rainbow trout, Oncorhynchus mykiss, and native Little Colorado spinedace, Lepidomeda vittata, a federally threatened cyprinid, in natural and experimental settings. In natural settings, we also examined occurrence and microhabitat use of two other native fishes, speckled dace, Rhinichthys osculus, and bluehead sucker, Catostomus discobolus. Native species co-occurred, as did rainbow trout and bluehead sucker. However, occurrences of Little Colorado spinedace and speckled dace were not significantly correlated with occurrence of rainbow trout. Total lengths of all three native species were significantly smaller at allopatric sites than at sites sympatric with rainbow trout. Microhabitat characteristics at sites with rainbow trout did not differ from those where the other three species were found, but did differ among the native species. In laboratory experiments with Little Colorado spinedace and rainbow trout, rainbow trout used the lower depth zone most, and spinedace increased use of the lower depth zone upon addition of rainbow trout. In addition, species tended to co-occur in zones, but used cover independently of one-another, suggesting a low level of agonistic interactions. However, after addition of a high density of rainbow trout, spinedace tended to use cover less than before. We suggest that the species can coexist at low rainbow trout densities. Potential negative effects of rainbow trout on Little Colorado spinedace likely increase with increasing densities of rainbow trout, and rainbow trout likely affect smaller size classes of Little Colorado spinedace more than larger ones.