The aim of this study was to investigate the process of glycerol catabolism in germinating Arabidopsis seed. A genetic screen was performed to isolate glycerol-insensitive (gli) mutant seedlings. Three separate mutant loci were identified (gli1, gli2 and gli3). Of these, only gli1 is unable to utilise glycerol. Following germination, gli1 seedlings transiently accumulate glycerol derived from the breakdown of storage oil and are more resistant to hyperosmotic stress, salt stress, oxidative stress, freezing and desiccation. Enzyme assays revealed that gli1 lacks glycerol kinase activity. GLI1 mapped to chromosome 1 near the putative glycerol kinase gene NHO1. Mutations in this gene were identified in three independent gli1 alleles. A cDNA encoding GLI1 was cloned and its function was proven by complementation of an Escherichia coli glycerol kinase (glpK) deletion strain. Quantitative RT-PCR analysis showed that GLI1 is expressed in all tissues, but is transiently upregulated during early post-germinative growth and leaf senescence. These data show that glycerol kinase is required for glycerol catabolism in Arabidopsis and that the accumulation of glycerol can enhance resistance to a variety of abiotic stresses associated with dehydration.