Purified chloroplasts (80% intact) with a photosynthetic activity of 195 μmol O2 mg chlorophyll−1 h−1 were prepared from the leaves of guayule. The activities of phosphoglyceromutase, enolase and pyruvate kinase were estimated in the purified chloroplasts and compared with those of Phaseolus radiatus Roxb. Activity of the pyruvate dehydrogenase complex (PDC) in the chloroplasts of guayule (448 nkat mg protein−1) was substantially higher than the PDC activity of Phaseolus (282 nkat mg protein−1). Incorporation of carbon from the added labelled carbon compounds, [14C]bicarbonate, [2-14C]pyruvate and [U-14C]-3-phosphoglycerate into isopentenyl diphosphate (IDP) was significant, indicating the ability of the chloroplasts to utilize Calvin cycle intermediates for IDP biosynthesis via acetyl CoA and mevalonate. DCMU inhibited the incorporation of 14CO2 and [14C]pyruvate into IDP. Acetoacetyl CoA thiolase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase activity were localized in the isolated chloroplasts. It is believed that the chloroplasts in the leaf of guayule are autonomous for the biosynthesis of IDP and this is presumed to be dependent on photosynthetic energy and carbon flow.