Background/Aims: This work is a study of the ability of three recombinant Zymomonas mobilis strains to release ice nucleators into their growth medium. Methods: The recombinant ice+Z. mobilis cells were tested for their ability to produce cell-free ice nucleators, under three different growth temperatures and three different glucose concentrations. Results: Cell-free ice nucleators were obtained from all the recombinant ice+Z. mobilis cells tested. The cell-free ice nucleation activity was not affected by the glucose concentration in the growth medium or the growth temperature. The freezing temperature threshold was below -7.6°C, demonstrating a class C nucleating structure of the ice nucleation protein. The size of the ice nucleators was less than 0.22 μm and their density was estimated as 1.024 ± 0.004 g/ml by Percoll density centrifugation. The properties of the detected ice nucleators, in addition to the absence of pyruvate decarboxylase activity in the spent medium (a cytosolic marker), support that the cell-free ice nucleation activity was due to the extracellular release of ice nucleators. Conclusion: These findings indicate that the recombinant ice+Z. mobilis cells could be valuable for future use as a source of active cell-free ice nucleation protein.