Amyloplasts have been purified from potato tubers by mechanical homogenization and gravity sedimentation through Nycodenz. Based on the recovery and latency of organelle-specific marker enzymes, the recovery of amyloplasts is approximately 13%, exhibiting 65% intactness, with less than 1% contamination by other subcellular fractions. These preparations were able to synthesize starch from glucose-1-phosphate plus ATP, or ADP-glucose but not from glucose-6-phosphate. Rates of starch synthesis from glucose-1-phosphate plus ATP were linear for up to 1 h and sensitive to the inhibitor 4,4-diisothiocyanato-stilbene 2,2-disulphonic acid (DIDS). Starch synthesis was optimal at pH 7.0 and was saturated by 5–10 mM glucose-1-phosphate and by 1 mM ADP-glucose. The results are discussed in the context of the pathway of starch synthesis and the transport of metabolites across the amyloplast envelope.