A class of plant growth regulators, gibberellins, induce the synthesis of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) in the aleurone layers of barley (Hordeum vulgare L. var. Himalaya) seeds. The purified alpha-amylase is composed of multiple isozymic forms with indistinguishable molecular weights, but different net charges. These alpha-amylase isozymes separate on isoelectric focusing gels into two groups, each containing multiple species. One group has an apparent isoelectric point (pI) of approximately 5.8 (the high pI group). The other group's pI values are around 4.5 (the low pI group). On some gels a small amount of protein focuses between the high and low pI isozymes. These proteins comigrate with the low pI isozymes upon reelectrophoresis. The synthesis of these two groups is temporally regulated. The high pI group is the dominant set of isozymes secreted from embryoless half seeds during the first two days of gibberellin administration. After four days, however, the major isozymes are those of the low pI group. This shift in isozyme pattern is due to a shift in their relative rates of synthesis. Peptide analysis of these two groups of isozymes with Staphylococcus aureus V8 protease and cyanogen bromide shows amino acid sequence differences. However, members within the same group have similar peptide patterns. Both groups of isozymes are synthesized in vitro in a wheat germ extract primed with poly(A)+ RNA isolated from gibberellin-treated aleurone layers. This indicates that the synthesis of the two groups of alpha-amylase isozymes is probably directed by two or more different populations of mature mRNA. A model that explains these observations and the available genetic information is that barley aleurone alpha-amylase isozymes are encoded by at least two sets of structural genes.