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We combine the use of dielectrophoretic positioning with electrical impedance measurements to detect and discriminate between individual bacterial spores on the basis of their electrical response. Using lithographically defined microelectrodes, we use dielectrophoresis to manipulate individual bacterial spores between the electrodes. The introduction of a single spore between the microelectrodes produces a significant change in electrical response that is species-dependent. When positioned between two electrodes and an AC voltage was applied, single spores caused current increases averaging 6.8 (+/-2.4) pA for Bacillus mycoides to 1.18 (+/-0.37) pA for Bacillus licheniformis. Using a mixture of spores of two different species, we demonstrate the ability to distinguish the species of individual spores in real time. This work demonstrates the feasibility of using impedance measurements for real-time detection and discrimination between different types of spores.