Objective: The aims of the study were to investigate the pattern of expression of the major subunits of the NF-κB transcription factor complex in human and rat heart development, and to characterise the timing of NF-κB activation by interleukin-1β (IL-1β) in rat neonatal cardiac myocytes. Methods: The expression of NF-κB subunits p65 and p50 and the inhibitory subunits IκB-α and IκB-β in human and rat myocardial samples was measured by immunoblotting, using antibodies specific to each subunit. The activation of NF-κB was measured in neonatal rat cardiac myocytes that were treated with IL-1β for different times (0–60 min). Depletion of the inhibitory factors IκB-α and IκB-β was assessed by immunoblotting. The presence of NF-κB DNA binding activity was measured directly in nuclear extracts by electrophoretic mobility shift assay (EMSA). Results: p65, p50, IκB-α and IκB-β are expressed at all stages of development analysed. In human myocardial samples, expression of p50, p65 and IκB-α show an apparent gradual decline relative to total protein. In contrast, the level of IκB-β remained relatively constant, suggesting a significant shift in the ratio of β and α subunits with development. In rat myocardium, p65, p50, IκB-α and IκB-β showed a gradual decline during development, with a particularly pronounced decrease between the ten day post-natal and adult samples. Treatment of neonatal rat cardiac myocytes with IL-1β (5 ng/ml) caused a rapid and transient depletion of IκB-α (reducing to 16±1.6% of initial levels within 5 min, returning to 82±10% within 60 min). A slower, less marked depletion is observed for IκB-β (24±6% by 30 min, returning to only 49±5% by 60 min). Rapid and transitory accumulation of NF-κB DNA binding activity was detected in the nucleus, with a pattern that correlated with the depletion of IκB-α. Conclusions: The principal NF-κB subunits p65, p50, IκB-α and IκB-β are present throughout development, suggesting that this transcription complex may participate in myocardial gene regulation throughout development and in the adult. Activation by IL-1β demonstrates that NF-κB probably plays a direct role in the regulation of gene transcription in response to cytokine activation in cardiac myocytes.