Objectives: N-terminal pro-B-type natriuretic peptide (NT-proBNP) is elevated in subarachnoid haemorrhage, brainstem death (BSD) and heart failure. We examined the relationship between NT-proBNP and cardiac functional status after BSD and left ventricular (LV) BNP precursor gene expression. Methods: We assayed NT-proBNP in the serum of potential heart donors investigated with pulmonary artery flotation catheters, transthoracic echocardiography and cardiac troponin (cTn) I and T. After 6.9 h of optimisation, haemodynamic studies were repeated to determine haemodynamic functional suitability for transplantation. Median (interquartile range (IQR)) NT-proBNP levels are reported according to initially measured dichotomised pulmonary capillary wedge pressure (PCWP), cardiac index (CI), indexed cardiac power output (CPOi), left ventricular ejection fraction (LVEF), wall motion score (WMS), extravascular lung water index (EVLWI), cTnT and cTnI and end-management functional suitability. LV biopsies were snap-frozen, mRNA extracted and reverse-transcribed, allowing performance of Taqman real-time polymerase chain reaction assays of mRNA–BNP precursor. Results: There were 79 subjects. Median NT-proBNP was 121 pg ml−1 (range 5–4139) and levels correlated with time from coning (p ≪ 0.01, r = −0.379). Higher NT-proBNP was found in donors with PCWP >14 mmHg; 504 (120–1544) versus 101 (38–285); p = 0.01; CI ≪2.4 l min−1 m−2 410 (123–1511) versus 95 (37–264); p = 0.001; CPOi ≪0.5 W m−2 256 (78–694) versus 105 (37–315); p = 0.02; LVEF ≪50% 231 (75–499) versus 72 (36–177); p = 0.04; WMS >2; 343 (80–673) versus 99 (37–236); p = 0.01; cTnT >0.1 μg ml−1 499 (127–967) versus 80 (36–173); p ≪ 0.001 and cTnI >1 mg ml−1 410 (97–684) versus 88 (36–190); p ≪ 0.01 and in hearts functionally unsuitable at end-optimisation; 189 (74–522) versus 85 (39–243); p = 0.02. Hearts functionally suitable for transplantation expressed significantly less mRNA encoding for BNP precursor (0.19-fold; p = 0.01). Conclusion: During or after BSD, NT-proBNP is released and the heart is a likely source. Higher NT-proBNP levels are associated with donor heart dysfunction and a failure to achieve haemodynamic functional suitability criteria. This supports the hypothesis that biomarkers, including NT-proBNP, may be useful in donor heart assessment.