A mathematical framework was developed to analyse rate limitation of non-steady-state photosynthesis following an increase in photon flux density (PFD). This analysis was employed to resolve an exponential phase of the photosynthetic response of Spinacia oleracea L. to a step increase from darkness to moderate PFD. This phase had a relaxation time of approximately 5 min, similar to the relaxation time for the activation of ribulose-1,5-bisphosphate carboxylase (Rubisco) as determined by freeze-clamp experiments following the same change in PFD. Furthermore, as the time in darkness prior to illumination was increased, the exponential phase contributed more to the overall trajectory of photosynthesis following the increase in light. The relaxation time for the increase in the contribution of this phase was 24 min. Freeze-clamp studies showed a relaxation time of 28 min for Rubisco deactivation in the dark. These results, together with measurements of RuP2 levels, suggest that the exponential phase resolved from gas exchange experiments was limited by activation of Rubisco and that Rubisco deactivation in the dark was the reason that this phase contributed more to the overall photosynthetic trajectory as time in darkness increased. A quantitative expression for the amount of extra photosynthesis that could have been obtained had Rubisco activated instantly following an increase in PFD was derived and discussed in relation to optimal functioning of the system under different environmental conditions.