Two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, were identified in two and one spots, respectively, in a proteome analysis of barley (Hordeum vulgare) seeds based on 2D gel electrophoresis and MS. HvTrxh1 was observed in 2D gel patterns of endosperm, aleurone layer and embryo of mature barley seeds, and HvTrxh2 was present mainly in the embryo. During germination, HvTrxh2 decreased in abundance and HvTrxh1 decreased in the aleurone layer and endosperm but remained at high levels in the embryo. On the basis of MS identification of the two isoforms, expressed sequence tag sequences were identified, and cDNAs encoding HvTrxh1 and HvTrxh2 were cloned by RT-PCR. The sequences were 51% identical, but showed higer similarity to thioredoxin h isoforms from other cereals, e.g. rice Trxh (74% identical with HvTrxh1) and wheat TrxTa (90% identical with HvTrxh2). Recombinant HvTrxh1, HvTrxh2 and TrxTa were produced in Escherichia coli and purified using a three-step procedure. The activity of the purified recombinant thioredoxin h isoforms was demonstrated using insulin and barley alpha-amylase/subtilisin inhibitor as substrates. HvTrxh1 and HvTrxh2 were also efficiently reduced by Arabidopsis thaliana NADP-dependent thioredoxin reductase (NTR). The biochemical properties of HvTrxh2 and TrxTa were similar, whereas HvTrxh1 had higher insulin-reducing activity and was a better substrate for Arabidopsis NTR than HvTrxh2, with a Km of 13 micro m compared with 44 micro m for HvTrxh2. Thus, barley seeds contain two distinct thioredoxin h isoforms which differ in temporal and spatial distribution and kinetic properties, suggesting that they may have different physiological roles.