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Context.—Whether cell membrane fatty acid (FA) composition is a useful indicator of vascular disease is unclear. Objective.—To study variation of erythrocyte (RBC) membrane FA in samples from healthy volunteers, hospitalized patients, and cardiac troponin I–elevated patients with myocardial damage without a priori assumptions as to FA composition. Design.—We separated FAs extracted from RBCs by gas chromatography and identified them by mass spectrometry. Fatty acids with abundance greater than 1% of total were quantified and compared: hexadecanoic (C16:0), octadecadienoic (C18:2), cis- and trans-octadecenoic (C18:1), and eicosatetraenoic (C20:4) acids. Deuterated standards established proportionality of FA recovery. The cis- and trans-C18:1 identification was verified by comparison with standards. Results.—In troponin-positive samples, C18:2 to C18:1 ratios were increased 30% compared with healthy controls or with random patient samples. Erythrocyte trans-C18:1 had a wide variation, ∼10-fold, in all groups but without differences between groups. Replicates showed that the wide range of RBC trans-FA load is not due to analytic variation. In healthy subjects, the RBC content of lower– molecular weight FAs (C16-C18) correlated with serum low-density lipoprotein cholesterol, but despite the established relationship between dietary trans-FA and increased low-density lipoprotein cholesterol, lipid profiles had no correlation with RBC trans-FA content. Conclusions.—Erythrocyte accumulation of unsaturated FA may be a useful indicator of vascular disease, whereas the wide range in trans-FAs suggests that both diet and genetic variation affect RBC trans-FA accumulation. Unsaturated FAs increase membrane fluidity and may reflect a natural response to subclinical vascular changes, which may in turn reflect increased risk of clinical disease.