The myocardial stress response to exercise is dependent on exercise intensity and thus understanding the molecular responses between various exercise intensity levels might aid in exercise prescription. Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor that mediates a variety of cellular processes including inflammation, immune responses, apoptosis and cell growth/development. NF-κB can be comprised of homo- and/or heterodimers formed from five distinct proteins: p50 (NF-κB1), p52 (NF-κB2), RelA (p65), c-Rel, and RelB. NF-κB is located in the cytoplasm and kept inactive by inhibitory proteins but following the exposure to a myriad of stimuli, an activated NF-κB dimer translocates to the nucleus and exerts transcriptional effects on upwards of 150 genes. To examine the activation of NF-κB in the myocardium following exercise, male Sprague-Dawley rats (n = 24) were exercised by treadmill running at 20 m/min for 30 min or 30 m/min for 20 min. At 0, 2, or 24 h following exercise, animals were anesthetized, hearts excised and immediately frozen in liquid nitrogen. Portions of hearts were homogenized, protein concentrations determined and extracts assayed for NF-κB activation (DNA binding activity) using electrophoretic mobility shift assays (EMSA). Visual examination of EMSA autoradiographs revealed an enhanced NF-κB activation in the hearts from exercised animals when compared with non-running controls. Subsequent supershift analyses using antibodies specific for NF-κB subunits showed the higher intensity exercise was associated with p65 (RelA) in the activated NF-κB complex while the NF-κB complex in hearts from animals exercised at the lower intensity was comprised primarily of p50. These data suggest exercise is capable of activating myocardial NF-κB and that a threshold for the activation of specific NF-κB subunits may exist.