Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 126.96.36.199; glycinamide ribonucleotide transformylase, EC 188.8.131.52; aminoimidazole ribonucleotide synthetase, EC 184.108.40.206; aminoimidazole carboxamide ribonucleotide transformylase, EC 220.127.116.11; and adenylosuccinate-AMP lyase, EC 18.104.22.168) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert  Arch Biochem Biophys 220: 179-187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin  Arch Biochem Biophys 224: 429-441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.