Unknown factors present in the serum of older adults impair lymphocyte function and may be responsible for anergy (absence of delayed-type hypersensitivity (DTH)) present in many older adults. Polyunsaturated fatty acids (PUFAs) and their metabolites are immunomodulatory and may play a role in clinical conditions of advanced age, including immune dysfunction. We hypothesized that PUFAs could be the factor(s) present in serum that contribute to impaired immune responses in older adults. Prior studies of serum PUFAs in older adults neither adequately control dietary PUFA intake, nor investigated the relationship of PUFAs and DTH responses. We determined serum PUFA concentrations in young adults with normal immune responses, and older adults with impaired (anergic elderly) or normal immunity (nonanergic elderly) before and after administering a standardized diet. After controlling for dietary intake, advancing age was associated with markedly higher serum concentrations of arachidonic acid (AA), dihomo-gamma-linoleic acid (DGLA), and eicosapentaenoic acid (EPA) and a lower AA:EPA ratio. Other serum PUFAs and the AA:DGLA ratio were unaffected by age. However, there was no difference between older adults with or without anergy. These data suggest advanced age is associated with marked alterations of serum PUFAs that are only apparent after strictly controlling dietary intake. However, there was no association of serum PUFA concentrations with DTH status among older adults.