Two container and substrate combinations were compared to determine which provided optimal survival of larvae of Chironomus tepperi and Glyptotendipes paripes in the absence of toxicants. Unfed final-stage larvae of G. paripes survived significantly (P < 0.05) better in waxed paper cups with sand substrate (92.8% after 3 days) than in glass tubes with a shredded paper substrate (85.3%). Survival of larvae of C. tepperi over the same period did not differ significantly in the 2 systems. Larvae of C. tepperi were bioassayed against 3 insecticides (technical and formulated imidacloprid, chlorpyrifos, and betacypermethrin) with both container and substrate combinations. Median lethal concentration values (24 h) obtained with waxed cups with sand were 1.8 times higher on average than those obtained with glass tubes with shredded paper (range 1.13-2.65 times). To determine the cause of this variability, solid-phase microextraction was used to measure changes in chlorpyrifos availability over time in the 2 bioassay systems. Chlorpyrifos concentrations in the waxed cups and sand system fell from 7.50 to 3.36 microg/liter over 24 h, probably as a consequence of chemical adsorption to the waxed surfaces. Chlorpyrifos concentrations in the glass tubes and paper system remained unchanged over this period. Excluding substrates from the containers had only a minor effect on chlorpyrifos availability. These results demonstrate that the behaviors of both test organisms and toxicants within bioassay systems need to be understood if the data generated with different systems are to be compared. Understanding how toxicant availability is affected by different container and substrate types is particularly important where bioassays are conducted with nominal concentration values rather than analytically determined exposure concentrations.