Understanding the Earth’s climate and its influencing factors is becoming increasingly important, yet the influence of aerosol particles on the climate is still uncertain. Numerous aerosols, such as dust, soot, and biological particles, can act as ice nuclei (IN) and trigger the freezing of supercooled liquid cloud droplets, thus changing cloud properties, the albedo, and the formation of precipitation. Ice nuclei of biological origin, like bacteria, fungi, or pollen, can have remarkably high onset freezing temperatures. Pummer et al. (2012) found that solubilized macromolecules are responsible for the ice nucleation activity of tree pollen and not the grains themselves. More recently, ice-nucleating macromolecules (INMs) have also been found on other tree tissues (Felgitsch et al., 2018). In general, INMs are present in much larger numbers than the micrometer-sized pollen grains and thus the emission of INMs from the biosphere might play a more important role than previously thought. Still, the chemical composition and structure of INMs remain largely unknown. To shine light on this, we extracted INMs from birch pollen with water. In order to concentrate the extracted INMs we used ice affinity purification, which enabled us to perform further characterization experiments with INM at varying concentrations. We found ice nucleation activity of birch pollen INMs at temperatures of up to -5°C, which is much higher than the -15°C previously reported by many studies. We further detected ice nucleation activity after filtration through a 10 kDa cutoff filter, which is much smaller than the previously reported 100 kDa. Lastly, we found the INMs to be sensitive to heat as the ice nucleation activity decreases with increased temperature and duration of heat treatments. Together with further analytics, like CD and fluorescence spectroscopy, this led us to the hypothesis that the INMs are made up of aggregates and might be proteinaceous. All our results especially the discovered ice nucleation activity at -5°C could necessitates a substantial revision of our view of tree pollen INMs as contributors to atmospheric ice nucleation.