A fourth molecular from of alpha-galactosidase, designated L(IV), an alkaline alpha-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-alpha-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K(m) determinations in McIlvaine buffer (200 millimolar Na(2)-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-alpha-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L(IV) was partially inhibited by Ca(2+), Mg(2+), and Mn(2+), more so by Ni(2+), Zn(2+), and Co(2+), and highly so by Cu(2+), Ag(2+), Hg(2+) and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-alpha-d-galactoside or by myo-inositol, but alpha-d-galactose was a strong inhibitor. As observed for most other forms of alpha-galactosidase, L(IV) only catalyzed the hydrolysis of glycosides possessing the alpha-d-galactose configuration at C(1), C(2), and C(4), and did not hydrolyze p-nitrophenyl-alpha-d-fucoside (alpha-d-galactose substituted at C(6)). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-alpha-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L(IV) in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L(IV)in vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L(IV) activity through coordination with some toxic cation introduced as a buffer contaminant.