Tissue culture techniques are widely used for gene transformation. However, regeneration of a transformed plant from the culture system depends on various factors, one of which is the plant hormone ethylene. Ethylene can have a positive or negative influence on callus formation and green plant regeneration depending on the genotype of donor plant, age of explant and stage of application. It is important to determine the stage at which controlling the amounts of ethylene would enable better regeneration. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and inhibitor aminoethoxyvinyl-glycine (AVG) were used in one-week treatments for a period of 10 weeks in barley (Hordeum vulgare L.) cultivar, Morex; only AVG was studied in 'Golden Promise'. The expression of ethylene biosynthesis and response genes (ACC synthase (ACS), ACC oxidase (ACO) and ethylene response1 (ETR1)) was also studied in cultured tissues from both cultivars and compared to other expression studies available from barley databases. Map locations of previously identified barley tissue culture quantitative trait loci (QTL) were compared to locations of expression-QTL (e-QTL) for ACS, ACO and ETR1 unigenes. In both cultivars, AVG treatments produced significant differences in amounts of ethylene produced. In Morex, regeneration rates were significantly improved in weeks 11-13 when compared to control. ACC treatment in Morex affected ethylene production but not the regeneration rates. The environmental conditions in which the donor plants were grown affected the performance of explants in culture. The ethylene biosynthesis and response genes belonged to multigene families. The genes were differentially expressed not only between the two cultivars, but also at different stages of tissue culture. Data mining results indicate that 14 of the 26 expression quantitative trait loci (QTL) for ACS, ACO and ETR1 unigenes could be mapped to 10 of the 13 tissue culture-related loci. The variation in expression can be exploited to develop methods to manipulate levels of ethylene to obtain increased regeneration from cultured systems.