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In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO₂ fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The k_cat was best fit by 22.3 ± 4.9 min⁻¹, the K_m for ATP by 0.104 ± 0.024 mm, and the K_i for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition …