Although many crustaceans have their pacemaking centre outside the heart in a cardiac ganglion, some very simple crustaceans are reported to have myogenic hearts. One such is the primitive planktonic freshwater crustacean Daphnia, whose single-chambered heart can beat over a wide range of frequencies. In evolutionary terms, Daphnids are intermediate between insects (arthropoda, hexapods) and more advanced crustaceans. Here we report changes in Daphnia heart with altered temperature and with application of caffeine. Intact Daphnia were immobilized within a small chamber of the stage on an inverted microscope and heart wall motion measured using a video edge detector (Crescent Electronics) or by analysis of digital images using ImageJ. Temperature in the chamber was measured with a bead thermistor. Daphnia heart rate was highly temperature dependent, falling from 320-330 min^-1 at room temperature (21°C) to around 50 min^-1 at 3°C. The rate of both contraction and relaxation also decreased dramatically. Filling and ejection fraction increased with reduced temperature. Occasional arrhythmias were seen on rapid cooling to 3-4°C. The addition of caffeine progressively slowed heart rate, by a maximum of around 20% at 15-20 mM caffeine. Caffeine-treated hearts showed a similar temperature-dependent decrease in heart rate as control hearts, but showed more arrhythmias on cooling. Arrhythmias were most common at 5-7°C in caffeine-treated hearts, and hearts that were not arrhythmic on cooling to 3°C often became transiently arrhythmic as they rewarmed slightly. The systems responsible for cardiac pacemaking in Daphnia remain unknown. In the tubular heart of Drosophila, recent gene-targeting studies implicate SERCA (Sanyal et al. 2006) and the twin-pore K⁺ (K2P) channel dORK1 (Lalevée et al. 2006) in pacemaking. Although Daphnia heart contains SR (Stein et al. 1966), the effects of caffeine reported here suggest the SR is not required for pacemaking. From the recently completed Daphnia pulex genome we have identified genes encoding SERCA, RyR, multiple K2P channels homologous to dORK1, and a single HCN channel homologue. Further studies will be required to identify which of these mechanisms regulate cardiac pacemaking in Daphnia.