In this study we developed an electrotransformation method for use with the Gram-positive bacterium Paenibacillus larvae-a deadly pathogen of honey bees. Combining multiple Bacillus electrotransformation methods to generate an initial protocol, we then optimized the following parameters for use with P. larvae: cell density of culture at harvest time, contents of the washing/electroporation solution, field strength of the electrical pulse, recovery growth medium, and recovery time period. With the optimized method, we achieved an average transformation efficiency of 1.9x10(5) transformants/mug DNA. The method is substantially different from the only other electrotransformation method for a Paenibacillus species found in the literature. This work should facilitate the study of the several previously discovered natural plasmids of P. larvae, and is a step toward developing a genetic system for this species.