Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 220.127.116.11). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO2-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 18.104.22.168). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.