The cyanobacterium Nostoc PCC 9229 forms an intracellular nitrogen-fixing symbiosis with the angiosperm Gunnera. In symbiosis the cyanobacterium is enclosed in darkness and receives carbon from the plant in an unknown form. Out of five putative plant carbohydrate sources tested in vitro, fructose and glucose were found to support nitrogen fixation in darkness. The other three dextrin, sucrose and Gunnera sp. mucilage could not induce nitrogenase activity in darkness. The stimulatory effect by fructose was also observed in illuminated samples. After four weeks incubation in darkness, nitrogenase was still active in cultures when fructose was added and multiple thick-walled nitrogen-fixing cells (heterocysts) were observed, and chlorophyll levels unchanged. The expression as shown by Northern blot analysis revealed that fructose influenced the gene expression of hetR, a gene necessary for heterocyst formation, in darkness. Fructose and glucose may therefore be the carbohydrates supplied by the host plant to induce heterocyst differentiation and nitrogen fixation in the cyanobiont Nostoc PCC 9229.