AbstractInsect cold hardiness is often mediated by low molecular weight cryoprotectants, such as sugars, polyols, and amino acids (AA). While many free‐living northern insects must cope with extended periods of freezing ambient temperatures (Ta), the ectoparasitic deer ked Lipoptena cervi imago can encounter subfreezing Ta only during a short autumnal period between hatching and host location. Subsequently, it benefits from the body temperature of the cervid host for survival in winter. This study investigated the cold tolerance of the species by determining its lower lethal temperature (100% mortality, LLT100) during faster and slower cold acclimation, by determining the supercooling point (SCP) and by measuring the concentrations of potential low molecular weight cryoprotectants. The LLT100 of the deer ked was approximately −16°C, which would enable it to survive freezing nighttime Ta not only in its current area of distribution but also further north. The SCP was −7.8°C, clearly higher than the LLT100, indicating that the deer ked displays freezing tolerance. The concentrations of free AA, especially nonessential AA, were higher in the cold‐acclimated deer keds similar to several other insects. The concentrations of proline increased together with γ‐aminobutyrate, arginine, asparagine, cystine, glutamate, glutamine, hydroxylysine, sarcosine, serine, and taurine. AA could be hypothesized to act as cryoprotectants by, e.g., protecting enzymes and lipid membranes from damage caused by cold. J. Exp. Zool. 317:1–8, 2012. © 2011 Wiley Periodicals, Inc.