Several Ascosphaera species were tested for their pathogenicity for larvae of the alfalfa leafcutting bee, Megachile rotundata. Larvae were reared aseptically on a sterile pollen-based diet and inoculated at 6 days of age with spores of the fungi. A. aggregata caused the distinctive syndrome called 'chalk brood'. Both A. apis and A. proliperda were pathogenic for larvae and produced syndromes similar to chalk brood in Apis mellifera. Chalk brood in M. rotundata was characterized by colour changes and sporulation of A. aggregata beneath the host cuticle. A. apis and A. proliperda caused no colour changes in infected larvae; white mycelia erupted through the cuticle before sporulation occurred. A. atra and A. major grew saprophytically on provisions of leafcutting-bee cells and did not cause larval disease. Spores taken from larvae killed by A. apis or A. proliperda grew less readily on the pollen diet, and appeared to be more virulent, than spores taken from subcultures.