Photoaffinity labeling with [(32)P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of (32)P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [(32)P] 8-N3ATP. (32)P-labeling was dependent upon ultraviolet irradiation and occurred with [(32)P] 8-N3ATP labeled in the α-position, indicating covalent incorporation of the photoprobe. Both [(32)P] 8-N3ATP and [(32)P] 8-N3GTP were incorporated to a similar extent into the 53-kilodalton (kDa) "large" subunit (LSu), but incorporation of [(32)P] 8-N3GTP into the 14-kDa "small" subunit (SSu) of RuBPCase was <5% of that measured with [(32)P] 8-N3ATP. Distinct binding sites for 8-N3ATP on the two subunits were indicated by different apparent K D values, 3 and 18 μM for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg(2+), anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N3ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [(32)P] 8-N3ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N3ATP binds to a site on the SSu that has physiological significance.