Ice nucleation was studied in field-fresh and acclimated (4°C without food for 11–20 days) samples of thespringtail Cryptopygus antarcticus Willem (Collembola, Isotomidae) at Rothera Research Station, Adelaide Islandon the Antarctic Peninsula. Ice nucleator activity (INA) was measured by a freezing droplet technique in additionto supercooling point (SCP) profiles and polyol, sugar, and water contents. Field and acclimated samplesshowed bimodal SCP distributions with a distinct high group (HG; mean SCP 28 to 210°C) and low group (LG:mean SCP 223 to 225°C), which were significantly different. Acclimation at 4°C increased the proportion of individualsin the LG relative to that in the HG without significant effects on the mean SCP of both groups. INA ofthe HG was significantly greater than that of the LG, and acclimation further reduced the INA of the LG. Thenumber of active ice nucleator agents (INAs) calculated for the HG of field samples increased by 23–100 timesover the temperature range 25 to 28°C compared to only 7 times for the LG over the same range. These differenceswere accentuated in the acclimation experiments. Glucose and galactose were the main carbohydrates inboth field and acclimated springtails, with the latter compound occurring in almost twice the concentration in theLG compared with that in the HG. Acclimation reduced the concentration of both compounds (glucose by 77%and galactose by 54%), whereas water content increased significantly. Digestion of food may have continued duringacclimation at 4°C, which could reduce the LG INA. Lowering of temperature over time is more likely toelicit a cold hardening response than constant temperature acclimation. INA numbers calculated at the nucleationtemperatures for C. antarcticus samples were higher in the LG than in the HG. However, inactivation of INAsmay be a key mechanism underlying cold hardening in this species, either by sequestration within the cellular matrixor by being only seasonally active.