The risk of Lyme disease for humans in the eastern United States is dependent on the density of host-seeking Ixodes scapularis Say nymphal stage ticks infected with Borrelia burgdorferi. Although many local and regional studies have estimated Lyme disease risk using these parameters, this is the first large-scale study using a standardized methodology. Density of host-seeking I. scapularis nymphs was measured by drag sampling of closed canopy deciduous forest habitats in 95 locations spaced among 2 degrees quadrants covering the entire United States east of the 100th meridian. Sampling was done in five standardized transects at each site and repeated three to six times during the summer of 2004. The total number of adults and nymphs of the seven tick species collected was 17,972, with 1,405 nymphal I. scapularis collected in 31 of the 95 sites. Peak global spatial autocorrelation values were found at the smallest lag distance (300 km) and decreased significantly after 1,000 km. Local auto-correlation statistics identified two significant high-density clusters around endemic areas in the northeast and upper Midwest and a low-density cluster in sites south of the 39th parallel, where only 21 nymphs were collected. Peak nymphal host-seeking density occurred earlier in the southern than in the most northern sites. Spatiotemporal density patterns will be combined with Borrelia prevalence data as part of a 4-yr survey to generate a nationwide spatial risk model for I. scapularis-borne Borrelia, which will improve targeting of disease prevention efforts.