The effects of two antibiotics, moxifloxacin, a fluoroquinolone, and erythromycin, a macrolide, on the HERG (human ether-a-go-go related gene) potassium channel were explored. Both of these antibiotics are known to have QT prolonging effects on the human ECG (Demolis et al. 2000; Mishra et al. 1999). The effects of these two drugs on both wild type and mutant heterologous HERG potassium currents (I_HERG) expressed in HEK or CHO cell lines were assessed using ‘whole-cell’ patch-clamp. Experiments were superfused at 37°C unless otherwise stated. Results are mean ± S.E.M. Both moxifloxacin and erythromycin blocked HERG in a concentration dependent manner. IC₅₀s were 54.9μM (n=5 for each of four drug concentrations) and 72.8μM (n=5 for each of 4 drug concentrations) for erythromycin and moxifloxacin, respectively. The time dependence of erythromycin block was further characterised. Cells were held at -100 mV and then subjected to a ‘short depolarisation’ (+40 mV for 10 ms). I_HERG tails were then observed at -40 mV (Milnes et al. 2003). Upon application of 60μM erythromycin, we observed 25 ± 6% block (n=5). The depolarisation was then lengthened to 200ms and 54 ± 3% block was seen (n=5, paired t test, P<0.01). These results are concordant with erythromycin either producing a rapid open-state-dependent block, or that the block includes a component of both open-state-dependent and closed-state-dependent block. Block by erythromycin was found to be strongly temperature dependent; 60μM erythromycin produced a block of 15 ± 9% (n=7) at 22°C compared to 51 ± 2% at 37°C (n=5, unpaired t test, P=<0.0001). Kirsch et al. (2004) found a similar temperature dependence for HERG blockade by erythromycin and suggested the reduction in block at room temperature could be due to the drug being much slower to cross the membrane and to access its binding site. The canonical HERG drug-binding site is thought to be located in the S6 domain. Two amino acid residues, F656 and Y652, are key constituents of this high affinity drug-binding site. Mutation of these residues to alanine only partially attenuated the HERG block by either moxifloxacin or erythromycin. These results provide further evidence for the existence of a second HERG drug-binding site distinct from the canonical S6 site.