Background We evaluated a new marker of heart transplant rejection, the breath methylated alkane contour (BMAC). Rejection is accompanied by oxidative stress that degrades membrane polyunsaturated fatty acids, evolving alkanes and methylalkanes, which are excreted in the breath as volatile organic compounds (VOCs). Methods Breath VOC samples (n = 1,061) were collected from 539 heart transplant recipients before scheduled endomyocardial biopsy. Breath VOCs were analyzed by gas chromatography and mass spectroscopy, and BMAC was derived from the abundance of C4-C20 alkanes and monomethylalkanes. The "gold standard" of rejection was the concordant set of International Society for Heart and Lung Transplantation (ISHLT) grades in biopsies read by 2 reviewers. Results Concordant biopsies were: Grade 0, 645 of 1,061 (60.8%); 1A, 197 (18.6%); 1B, 84 (7.9%); 2, 93 (8.8%); and 3A, 42 (4.0%). A combination of 9 VOCs in the BMAC identified Grade 3 rejection (sensitivity 78.6%, specificity 62.4%, cross-validated sensitivity 59.5%, cross-validated specificity 58.8%, positive predictive value 5.6%, negative predictive value 97.2%). Site pathologists identified the same cases with sensitivity of 42.4%, specificity 97.0%, positive predictive value 45.2% and negative predictive value 96.7%. Conclusions A breath test for markers of oxidative stress was more sensitive and less specific for Grade 3 heart transplant rejection than a biopsy reading by a site pathologist, but the negative predictive values of the 2 tests were similar. A screening breath test could potentially identify transplant recipients at low risk of Grade 3 rejection and reduce the number of endomyocardial biopsies.