The hyperpolarization-activated cation current I (f) plays a key role in the modulation of rhythmic activity in cardiac pacemaker cells and spontaneously firing neurons. I (f) is generated by hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-HCN4) and comprises two components: the fast instantaneous current (I (INS)) and the slowly developing steady-state current (I (SS)). We found that in I (f) traces evoked by consecutive hyperpolarization, the I (INS) amplitude of the second trace was up to 50% larger than the first. I (SS) was identical. This pre-hyperpolarization mediated enhancement of I (INS) was maximal in channels displaying slow kinetics (sinoatrial I (f), HCN3, and HCN4), while it was almost negligible for fast channels (HCN1 and HCN2). The enhancement quantitatively correlated with the frequency of hyperpolarization. Analysis of HCN4 currents suggested that enhancement was facilitated by incomplete deactivation, confirmed by HCN2-HCN4 chimeric studies. It is important to note that intracellular Cl(-) was found to be a cellular suppressor of I (INS) enhancement. Cl(-) inhibited the enhancement with an IC(50) around 25 mM and Hill coefficients between 2 and 6. Cl(-) shifted V (0.5) by +7 mV when [Cl(-)](i) was increased from 11 to 141 mM. In conclusion, I (INS) represents a quantitatively important component of I (f) at low Cl(-) (as found in most cell types). Moreover, an increase in cellular Cl(-) will suppress enhancement of I (INS) and, hence, potentially affect the electrical properties of cells.