The primary aims of this study were to quantify the density of Ixodes pacificus Cooley and Kohls nymphs and adults of the same generational cohort collected within a single year in six oak or madrone leaf litter habitats and to compare the prevalence of infection with Borrelia burgdorferi sensu lato (s.l.) in adults originating from nymphal cohorts with a low (<1%) versus high (>10%) infection prevalence. Because adult densities were very low both in and adjacent to several sites, direct comparisons of infection prevalence between nymphs and adults were possible only for two sites. Mean density in these sites decreased from 11.95/100 m2 for nymphs to 0/100 m2 for adults in leaf litter, and infection prevalence with B. burgdorferi s.l. was four-fold higher in nymphs (7.4%) versus adults (1.6%) of the same generational cohort collected in ecotones bordering the leaf litter areas. Assuming a density of adults in leaf litter of 0.04/100 m2 (mean for all six examined sites) and an infection prevalence similar to that found in adults collected from litter ecotones, the risk of encountering infected ticks in leaf litter decreased >1,000-fold from the nymphal to adult stage. Regardless of site-specific infection prevalence in the nymphal stage (n = 2 sites; 0.7 versus 14%), the infection prevalence for the adults of the same generational cohort was similarly low (1.5-1.6%). Peak densities of adult I. pacificus were 0-0.1/100 m2 in leaf litter, 0-6.5/100 m2 in ecotonal grasslands, and 2.0-39.0/100 m2 in ecotonal chaparral. Despite more intensive sampling efforts in leaf litter, the vast majority of the 282 adults collected came from grass or chaparral ecotones (98.9%, n = 279) rather than leaf litter (1.1%, n = 3). The study yielded eight B. burgdorferi s.l.-infected adults; four of these carried B. burgdorferi sensu stricto Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, and the remaining four were infected with currently undescribed B. burgdorferi s.l. spirochetes. This is the first study comparing both density of I. pacificus nymphs and adults and prevalence of infection with B. burdorferi s.l in these ticks within the same generational cohort and sampling area.