3-Phosphoglycerate (PGA)-dependent O(2) evolution by mesophyll chloroplasts of the C(4) plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of O(2) evolution. The extent of inhibition of O(2) evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophyll, concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA.Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent O(2) evolution. Protection against this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, if the chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorganic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent O(2) evolution by isolated chloroplasts of the C(3) plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C(4) chloroplasts.Oxalacetate-dependent O(2) evolution and its stimulation by the uncoupler, NH(4)Cl, were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C(4) crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA.