Chilling injury is sustained by dry pollen of Typha latifolia L. upon hydration in germination medium at 0 degrees C. This injury is evidenced as poor germination, low vigor, and depressed respiration. Isolated mitochondria showed multiple sites of impaired electron transport. Besides losses of cytochrome (Cyt) c and NAD(+), the activities of membrane-bound enzyme complexes such as Cyt oxidase, NADH-duroquinone oxidoreductase, succinate-duroquinone oxidoreductase, and malate-duroquinone oxidoreductase were severely affected.Similarly, as in isolated mitochondria, in situ tests of mitochondrial activity showed that Cyt c was partially lost from its site of action. Re-addition of the lost Cyt c to the grains restored the N,N,N',N'-tetramethyl p-phenylenediamine dihydrochloride plus ascorbate-mediated electron transport from Cyt c to O(2), but did not significantly accelerate the overall O(2) uptake. Electron flow to duroquinone in the injured grains was low, indicating that lesions at the substrate side of ubiquinone determine the rate of O(2) consumption. Leakage of NAD(+), and also of adenylate phosphates and Krebs cycle substrates out of the injured grains, was considerable.Increasing the initial moisture content of the grains strongly enhanced their resistance to cold hydration. Below 17% moisture content (fresh weight basis), the decrease in vigor closely matched the loss of NAD(+) and adenosine phosphates. Vitality was irreversibly lost by cold hydration below 10 to 12% initial moisture content.Injury to dry pollen was prevented by imbibition at 27 degrees C. Decrease of vigor and increased leakage, however, started below 20 degrees C, and complete loss of vitality occurred below 10 degrees C.These results are interpreted as evidence that loss of membrane integrity is the primary cause of imbibitional chilling injury.