The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system can generate spontaneous electrical activity. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker “funny” current (If) during diastolic depolarisation (Choudhary et al, 2015). HCN4 channels localise to lipid rafts and disorganisation of rafts results in redistribution of the channels, altering their kinetic properties (Barbuti et al, 2004). S-palmitoylation regulates key cardiac Na+ and Ca2+ handling proteins, influencing their membrane microdomain localisation and function (Howie et al, 2018; Reilly et al, 2015) and acting as a mechanism of targeting transmembrane proteins and transporters into lipid rafts (Gök et al, 2020).  This in vitro study was conducted to investigate HCN4 palmitoylation and its functional consequences. Resin assisted capture (acyl-RAC) of acylated proteins was used to assess palmitoylation of full length HCN4 and its intracellular amino and carboxyl termini fused to YFP in human embryonic kidney (HEK) cells as well as endogenously expressed HCN4 in neonatal rat ventricular myocytes. Site-directed mutagenesis using In-Fusion Cloning was used to construct alanine mutations of the palmitoylation sites. Whole-cell patch-clamp recordings from stable wild-type (WT) and mutant C93A/C179A HCN4-expressing HEK 293 cells employed an internal solution comprised of (in mmol/L): 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, and 10 HEPES (titrated to pH 7.2 with KOH) and external solution comprised of (in mmol/L): 140 NaCl, 4 KCl, 2.5 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (titrated to pH 7.4 with NaOH). Phylogenetic analysis was used to explore the evolutionary emergence of HCN4 palmitoylation within the pre-metazoan and metazoan lineage. Acyl-RAC (n = 5) established that palmitoylation of full length HCN4 occurs. Mutagenesis of intracellular cysteines at the N-terminus revealed that C93 and C179 are both sites of HCN4 palmitoylation (n = 3). A double cysteine-to-alanine mutation C93A/C179A of full length HCN4 resulted in an ∼ 77% reduction (0.52 ± 0.05 for WT vs 0.12 ± 0.06 for C93A/C179A; mean ± SEM, n = 3; unpaired t test; P < 0.01) in palmitoylation in comparison to wild type HCN4. Under whole-cell patch clamp, removal of the two N-terminal palmitoylation sites did not significantly alter the half maximal activation voltage (V0.5: -82.4 ± 5.20 mV for WT vs -82.8 ± 1.15 mV for C93A/179A; mean ± SEM, n = 3 and 9 respectively; unpaired t test; P = 0.91) but altered the activation slope factor (k: 6.21 ± 0.17 mV for WT vs 10.54 ± 0.64 for C93A/C179A; n = 3 and 9; unpaired t test P < 0.05).  Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. A polybasic cassette potentially driving the palmitoylation of cysteine 93 in human HCN4 was conserved widely across all classes of HCN4 vertebrate orthologs. Collectively, the work in this study provides clear evidence for N terminal palmitoylation of the HCN4 channel.