Streptonigrin, an antibiotic with antineoplastic activity, inhibited rat liver phosphoenolpyruvate carboxykinase with an I50 of 0.3 microM when excess FeCl2 was present. No inhibition occurred in the absence of added metal ion. Inhibition was partial and noncompetitive versus ITP and oxalacetic acid. The enzyme was more susceptible to inhibition by streptonigrin in the absence of substrates. Fe2+ supported inhibition by streptonigrin to a greater extent than did Fe3+, while Mn2+ activated the enzyme in the presence of streptonigrin. For maximum inhibition, at least a 3-fold molar excess of iron over streptonigrin was required. The methyl ester of streptonigrin was also an inhibitor (I50 = 4 microM) while the fragment containing the C and D rings was not, indicating that inhibition did not depend solely on the presence of the picolinic acid moiety. When oxalacetate synthesis was measured, streptonigrin plus iron had no more effect on enzymatic activity than iron alone, and Mn2+ was capable of stimulating the streptonigrin-Fe2+ inhibited enzyme.