SummaryThe incidence of chalk brood infection was compared among groups of 10 honeybee (Apis mellifera) colonies maintained on each of 5 different comb types: comb drawn from foundation, honey-super comb that had been used for storing honey but not for brood rearing, comb that had been used as brood comb for 5–30 and 30–45 years, and old brood comb that had been fumigated with ethylene oxide prior to use. The level of chalk brood disease ranged from negligible to over 6% and was lowest in the colonies with foundation or super comb, intermediate in the colonies with fumigated comb, and highest in the colonies with the two types of older brood comb. The results indicate that old brood comb contributes to the development of chalk brood disease in honeybee colonies, possibly by serving as a reservoir forAscosphaera apisspores or by promoting the growth of the pathogen.