The site of ice nucleation in the freeze-tolerant, sub-Antarctic beetleHydromedion sparsutumhas been investigated. Ice+bacteria, active at above −2.0°C, were isolated from the guts of beetles and identified as a fluorescentPseudomonasspecies. Other possible sites of nucleation, including the hemolymph, were examined but had a lower activity. Ice+bacteria were isolated from mixed populations, isolated from the guts of adult beetles, and grown on nutrient agar plates and in nutrient broth. Nucleation activity of the broth culture peaked after only 2 days although the number of live cells continued to increase until day 6. These cultures were used to determine the maximum nucleation activity of a bacterial suspension in sterile distilled water (−3.4°C) and the dilution factor required to cause a 50% reduction in activity (104). The original bacterial suspension had an absorbance of 0.5 measured at 660 nm and contained 6 × 1011bacteria per milliliter. From this it is estimated that only 1 in 106bacteria possessed the highest levels of ice-nucleating activity. Other insect species, including Perimylops antarcticus,which are found in habitats similar to that ofH. sparsutum,were examined for the presence of ice+bacteria. All contained ice-nucleating bacteria in their guts but with a lower level of activity than in H. sparsutum.