Behavioral bioassays remain a standard tool in the discovery, development, and registration of arthropod repellents. Tick repellent bioassays are generally uncomplicated, but their results can be affected by basic variables (e.g., dimensions of testing materials, substrate, timing, temperature) of the assay. Using lone star tick, Amblyomma americanum (L.), nymphs in climbing bioassays, we tested for the effects of substrate, solvent, and drying time on tick responses. In dose-response tests, the widely used repellents N,N-diethyl-3-methyl benzamide (deet) and 1-methyl-propyl-2-(hydroxyethyl)-1-piperidinecarboxylate (picaridin) were applied to filter paper strips and challenged by ticks at 10, 20, 30, 40, and 120 min after application. At 10-min drying time, repellency at the intermediate concentration 500 nmol repellent/cm2 filter paper was significantly lower for ethanol solutions of deet and picaridin (0 and 10% ticks repelled, respectively) than for solutions of deet and picaridin in acetone (96.7 and 76.7% ticks repelled, respectively). Repellency was greatest for both the acetone and ethanol solutions of deet and picaridin when challenged 120 min after application, and at shorter drying times at the highest concentration tested (2,000 nmol compound/ cm2). The repellency of picaridin relative to deet differed at some combinations of solvent and drying time but not others. In dose-response tests using different paper substrates and a drying time of 10 min, both ethanol and acetone solutions of deet differed in repellency, depending on both the paper substrate and the solvent. However, there were no differences in repellency between ethanol and acetone solutions of deet applied to nylon organdy in an in vitro and in an in vivo (fingertip) bioassay. When deet in solution with various proportions of ethanol:water was applied at 2,000 nmol deet/cm2 filter paper, the proportion of ticks repelled decreased as the proportion of water in the test solutions increased. Somewhat similar results were seen for solutions of deet in an acetone solvent. Water absorbed from the atmosphere may affect the efficacy of repellents in solution with anhydrous ethanol. Overall, results obtained from bioassays that differ in seemingly minor ways can be surprisingly different, diminishing the value of comparing studies that used similar, but not identical, methods. Nylon organdy or another similar thin cloth may be preferable to filter papers and copier paper for minimizing solvent-related differences. When a paper substrate is used, acetone may be the more suitable solvent if the solubility of the test compound and other factors allow.