The heat production of seeds during germination comes from metabolism as well as hydration. Previous studies either lack continuous measurements, or are based on samples composed of more than one seed, thus failing to characterize differences among the critical phases of germination. This study examines the potential of isothermal calorimetry to characterize water uptake and metabolism in single seeds. Seeds of malting barley (Hordeum vulgare L.) and winterfat [Krascheninnikovia lanata (Pursh) A.D.J. Meeuse & Smit], two species with contrasting seed size, structure, composition and selection history, were used to determine patterns of heat production rate by isothermal calorimetry during water uptake and germination. Embryos of malting barley contributed less than 4% of total seed weight, and metabolic heat production during Phase I of germination was negligible compared to that due to hydration. Embryos accounted for 74% of seed mass for winterfat, and the majority of heat produced in Phase I was due to metabolic heat release. The total heat production rate in Phase I decreased rapidly in malting barley due to slowing of hydration reactions, but increased gradually in winterfat due to an increasing metabolic rate. The heat production rate at the end of Phase II was about twice as high in malting barley as in winterfat. This indicates a higher metabolic activity for malting barley than for winterfat seeds during germination, which may have also contributed to the rapid increase in the heat production rate of malting barley seedlings during Phase III, compared to the gradual increase in heat production rate of winterfat. The comparison between excised embryos and intact seeds indicates that the covering tissues delay radicle emergence in malting barley, but not in winterfat, due to differences in seed structure between the two species.