Red pine ('Pinus resinosa' Ait.) has been planted extensively throughout southern Ontario since the 1920s to restore marginal land to productive use, prevent soil erosion, conserve water resources, and provide timber, educational opportunities and recreation. In recent years, unprecedented rates of mortality in maturing plantations are challenging forest managers in their attempts to adhere to traditional management strategies and to realize expected economic benefits. Although earlier studies linked mortality with various causes, no comprehensive evaluation of red pine mortality was undertaken. This study investigated abiotic and biotic factors associated with pocket mortality at 12 plantations in southern Ontario. Trenches, 2 m deep, were dug in diseased and healthy areas of nine plantations exhibiting pocket mortality, and in three disease-free plantations. Below-ground assessments of soil physical characteristics and rooting were combined with above-ground measurements to compare healthy and diseased sites, plots and trees. Root disease pathogens, ('Armillaria ostoyae' and 'Heterobasidion annosum'), black pineleaf scale ('Nuculaspis californica'), drought and iron deficiency were associated with mortality. Rooting depth was greater on healthy sites than on mortality sites, but there was no significant difference between healthy and diseased plots on mortality sites, nor between living and dead trees in mortality plots, except on Armillaria root disease sites where living trees rooted more deeply than trees that recently died. The most notable and consistent difference between healthy and diseased sites was the alkaline C horizon of disease sites compared with the acidic C horizon of healthy sites. Armillaria root disease was the single most frequent biotic factor associated with decline and the rDNA 5S gene and IGS2 region of several ' Armillaria' spp. were sequenced, and an identification protocol was developed based on novel primers and RFLP analyses. RAPD and ISSR analyses of 'A. ostoyae' isolates from two sites provided evidence of carryover of 'Armillaria' from the original forest, through the agricultural period, to the current red pine plantation, as well as of recolonization of an area from which 'Armillaria' had presumably been removed through cultivation. An experiment investigating the contributions of soil type and genetics to rhizomorph production by 'A. ostoyae' isolates supported a greater inherent genetic component than from soil effects.