We measured three components of the behavioral activity of blacklegged tick (Ixodes scapularis Say) nymphs under controlled conditions of daylength, temperature, and relative humidity in the laboratory. Temperature and relative humidity were experimentally manipulated among replicated treatment groups in two experiments. Humidity treatment had a significant effect on mean questing height, which was greater at 100% RH than at any lower humidity (P < 0.01), but it had no effect on mean distance moved between observations or the mean percentage of time in questing posture. Temperature had no effect on mean questing height, but significantly affected mean distance moved (P < 0.01) and the percentage of time in questing posture (P < 0.001). Mean distance moved and percentage of time in questing posture were both greater at 25°C than at higher or lower temperatures. A mechanistic understanding of the factors affecting tick behavior is likely to be useful for interpreting field data, designing field studies, and predicting risk of exposure to tick-borne pathogens.