The focal distribution of Ixodes scapularis Say and Amblyomma americanum (L.) was measured at 10-m intervals across the 1-ha study area placed within a mixed hardwood-pine forest with a sparse to dense shrub layer. At each of the resulting 100 locations, 1-m² plots were established and, following characterization of vegetation and edaphic conditions, were periodically flagged throughout the nymphal tick season. I. scapularis and A. americanum nymphs appeared to be nonrandomly distributed within the forested study area. Although only a few I. scapularis nymphs were collected overall, several locations consistently yielded more A. americanum nymphs than the remaining sites. I. scapularis was found in plots with substantial shrub density, extensive leaf litter, and deep soil duff layers, all of which contribute to creating high-humidity conditions for questing ticks. Questing A. americanum were more frequently encountered in more open canopy situations with less dense shrub layers and significantly reduced litter and duff layers. A. americanum appears to be more tolerant of desiccating conditions and so is able to occupy microsites or microhabitats that are less favorable for sympatric I. scapularis. The implications of these findings on the ability to sample ticks and estimate disease transmission risk are discussed.