We conducted a series of experiments to determine if extraradical hyphae of VAM fungi acclimate to low temperatures. Blocks of field soil containing VAM fungi were either slowly cooled or held at room temperature prior to freezing. Infectivity of VAM fungi was greater in soil that was slowly cooled. We hypothesized that greater infectivity following freezing resulted from cold acclimation of extraradical hyphae. This hypothesis was tested using in vitro mycorrhizas cultured in two-compartment Petri plates, in which hyphae grew into a separate compartment. Metabolic activity of these hyphae following freezing was assessed using a vital stain. The majority of cultures that were slowly cooled prior to freezing contained active hyphae, whereas hyphal activity was almost completely eliminated by freezing in non-precooled cultures. Freezing temperature influenced survival and activity of hyphae. To our knowledge this is the first report of acclimation to cold temperatures by VAM fungi.