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Primer extension was used to characterize alpha-amylase mRNAs from aleurone tissue of barley (Hordeum vulgare L. cv. Himalaya) grains. Two synthetic oligonucleotides,specific for the low-pl and high-pl alpha-amylase groups, were used as primers for synthesis of cDNAfrom total RNA preparations. Between them, these two oligonucleotides appear to account for all major alpha-amylase mRNAs as judged by hybrid-arrested translation of alpha-amylase mRNAs in a cell-freesystem. Reconstruction experiments indicated that the levels of extendedprimers (determined by scintillation counting) were directly proportional to the level of input mRNAover a wide range. This indicates that the technique is suitable for quantification of relative levelsof individual alpha-amylase from approximately 2% to 100% of maximal levels. The nucleotide sequences of extended primers defined twodifferent alpha-amylase mRNAs in each of the low-pl and high-pl groups, and possibly a third mRNA in thehigh-pl group.