Burrowing mayflies (Hexagenia bilineata) were analyzed to assess longitudinal patterns in their cadmium and mercury content along the upper Mississippi River. Emergent mayflies (subimagoes and imagoes) were collected in 1988 at 34 sites (25 navigation pools), extending 1260 km from Little Falls, Minnesota, downstream to St. Louis, Missouri. Cadmium concentrations in composite samples of whole mayflies ranged from 7 to 219 ng/g dry weight in females and from <7 to 265 ng/g in males. Cadmium concentrations were highest (~> 150 ng/g) in samples from Pools 2 and 3 (downstream from the Twin Cities metropolitan area), 15 (near the Quad Cities metropolitan area), and 27 (near the St. Louis metropolitan area). Cadmium concentrations in female mayflies decreased significantly with distance downstream from Pool 2 at river mile 825 to Pool 9 at river mile 648, paralleling spatial trends in the cadmium contamination of sediments in the reach downstream from the Twin Cities metropolitan area, reported in earlier studies; cadmium burdens in mayflies followed a similar spatial trend. Concentrations of mercury were much less variable, ranging from 44 to 102 ng/g dry weight in female mayflies and from 60 to 177 ng/g in males; concentrations and burdens were highest in mayflies collected at Pools 2, 20, 22, 25, and 27. Mercury concentrations in females decreased significantly with distance downstream from Pool 2 at river mile 825 to Pool 5A at fiver mile 728.5. Concentrations of mercury and cadmium in composite samples of female mayflies were not correlated, indicating dissimilar longitudinal patterns in concentrations of the two metals. Concentrations and burdens of both metals varied significantly between males and females; consequently, we recommend that programs involving analyses of mayflies to survey or monitor metals in aquatic systems analyze separately males and females.