Ticks and tick-borne diseases are of increasing concern in the United States, and understanding tick behavior could be crucial to limiting the exposure of humans and other animals to ticks. Here, we wanted to understand the drivers of questing and burrowing in Amblyomma americanum and A. maculatum. We used a factorial design to assess the effects of substrate (sand, soil, and rock), temperature (16° and 22° C), tick species (A. maculatum and A. americanum), and competition (homogenous or heterogenous mixture of ticks in the arena) on tick behavior in laboratory arenas. Questing varied significantly between tick species, with 61% of A. americanum questing compared to 35% of A. maculatum. Substrate similarly affected questing, with most ticks questing in the arenas with sand (83%) compared to arenas with rock (35%) or soil (25%). Burrowing behavior was also influenced by substrate; every tick in the arenas with rock (100%) and nearly all in the arenas with soil (98%) exhibited burrowing, whereas only 10% burrowed in sand. A. americanum traveled longer distances than A. maculatum (23 ± 2.8 cm vs 9.7 ± 2.3 cm). The disparity in laboratory behavior between sand and rock/soil substrates might warrant further consideration in a field setting, as understanding factors that inhibit host-seeking behaviors of ticks can inform management of public spaces and potentially help mitigate tick-exposure.