The influence of hourly fluctuating irradiance on a continuous culture ofChlorella vulgaris on the quantum yield for stable charge separation at photosystem II (ϕII,E) was examined. Linear regression analysis between ϕII,E and Ft or Fm' showed that Ft or Fm' explained 86 and 54% of the variability in ϕII,E respectively. The total amount of reducible QA remained constant during the light period. Only pigments that are known to operate in the xanthophyll cycle were variable. Violaxanthine reversibly decreased during the day, while an opposite pattern was observed for antheraxanthine. The changes in violaxanthine significantly (P ≪ 0.001) explained 87% of Fm' and 90% of the estimated values for the rate constant for energy dissipation by non-photochemical pathways. The magnitude of the variability in ϕII,E was well within range of regulation by the photosynthetic processes itself. The results suggest that ϕII,E alone can not be used as an index for the optimal cultivation of microalgae. Guidelines are proposed to translate fluorescence-derived parameters into general strategies to optimize productivity. Estimates ofin situ rates of primary photosynthesis derived from ϕII,E or from P/I derived estimates were significantly different, while predicted concentrations of carbohydrate concentrations were similar.