Raffinose family oligosaccharides (RFO) accumulate in many developing seeds and are degraded during seed germination. However, acidic α-galactosidase (AGAL) activity and subcellular location do not correlate with raffinose depletion; alkaline α-galactosidases (AGA) may be responsible for RFO hydrolysis in germinating seeds. Three cDNA clones for AGA/SEED IMBIBITION PROTEIN were obtained from the Pioneer Hi-Bred maize expressed sequence database. Two of the clones were expressed in Escherichia coli, and the recombinant proteins, when incubated with naturally occurring galactosides or p-nitrophenyl α-d-galactose, exhibited AGA activity with maximum catalysis at pH 7.5 (ZmAGA1) or pH 8.5 (ZmAGA3). No raffinose biosynthetic capacity was observed with either enzyme. Maximal α-galactosidase activity in mature dehydrated, germinating and germinated maize (Zea mays) seeds occurred at pH 7.5. ZmAGA1 was the sole family member detected in seeds and maize Hi-II, embryo-derived, callus cells. Its transcript accumulated when seed germination was interrupted by heat, cold or dehydration stress, but not in response to NaCl. Tissue prints localized transcripts to the scutellum or the embryo axis, depending on the stress applied. In maize Hi-II callus cells, transcripts accumulated when callus was subjected to heat stress (42 °C), during which ZmAGA1 transcript accumulation was further induced by sucrose. Galactosides in a variety of forms, including raffinose, partially repressed the sucrose-induced accumulation of transcript in heat-stressed callus.