INTRODUCTION Despite the evidence that the hyperpolarization-activated current (If) is highly modulated in human cardiomyopathies, no definite data exist in chronic atrial fibrillation (cAF). We investigated the expression, function, and modulation of If in human cAF. METHODS AND RESULTS Right atrial samples were obtained from sinus rhythm (SR, n = 49) or cAF (duration >1 year, n = 31) patients undergoing corrective cardiac surgery. Among f-channel isoforms expressed in the human atrium (HCN1, 2 and 4), HCN4 mRNA levels measured by RT-PCR were significantly reduced. However, protein expression was preserved in cAF compared to SR (+85% for HCN4); concurrently, miR-1 expression was significantly reduced. In patch-clamped atrial myocytes, current-specific conductance (gf) was significantly increased in cAF at voltages around the threshold for If activation (-60 to -80 mV); accordingly, a 10-mV rightward shift of the activation curve occurred (P < 0.01). β-Adrenergic and 5-HT4 receptor stimulation exerted similar effects on If in cAF and SR cells, while the ANP-mediated effect was significantly reduced (P < 0.02), suggesting downregulation of natriuretic peptide signaling. CONCLUSIONS In human cAF modifications in transcriptional and posttranscriptional mechanisms of HCN channels occur, associated with a slight yet significant gain-of-function of If , which may contribute to enhanced atrial ectopy.