Three isoforms of alpha-glucosidase (EC 3.2.1.20) have been extracted from pea (Pisum sativum L.) seedlings and separated by DEAE-cellulose and CM-Sepharose chromatography. Two alpha-glucosidase isoforms (alphaG1 and alphaG2) were most active under acid conditions, and appeared to be apoplastic. A neutral form (alphaG3) was most active near pH 7, and was identified as a chloroplastic enzyme. Together, the activity of alphaG1 and alphaG2 in apoplastic preparations accounted for 21% of the total acid alpha-glucosidase activity recovered from pea stems. The vast majority (86%) of the apoplastic acid alpha-glucosidase activity was due to alphaG1. The apparent K(m) values for maltose of alphaG1 and alphaG2 were 0.3 and 1.3 millimolar, respectively. The apparent K(m) for maltose of alphaG3 was 33 millimolar. The respective native molecular weights of alphaG1, alphaG2, and alphaG3 were 125,000, 150,000, and 110,000.