Blue light has many direct and indirect effects on photosynthesis. The impact of blue light on mesophyll conductance (g(m)), one of the main diffusive limitation to photosynthesis, was investigated in leaves of Nicotiana tabacum and Platanus orientalis, characterized by high and low g(m), respectively. Leaves were exposed to blue light fractions between 0% and 80% of incident light intensity (300 micromol photons m(-2) s(-1)), the other fraction being supplied as red light. Leaves exposed to blue light showed reduced photosynthesis and unaltered stomatal conductance. The g(m), measured using the chlorophyll fluorescence-based method, was strongly reduced in both plant species. Such a reduction of g(m) may not be real, as several assumptions used for the calculation of g(m) by fluorescence may not hold under blue light. To assess possible artefacts, the electron transport rate measured by fluorescence (J(f)) and by gas-exchange (J(c)) were compared in leaves exposed to different fractions of blue light under non-photorespiratory conditions. The two values were only equal, a prerequisite for correct g(m) measurements, when the illumination was totally provided as red light. Under increasing blue light levels an increasing discrepancy was observed, which suggests that J(f) was not correctly calculated, and that such an error could also upset g(m) measurements. Blue light was not found to change the absorbance of light by leaves, whereas it slightly decreased the distribution of light to PSII. To equate J(f) and J(c) under blue light, a further factor must be added to the J(f) equation, which possibly accounted for the reduced efficiency of energy transfer between the pigments predominantly absorbing blue light (the carotenoids) and the chlorophylls. This correction reduced by about 50% the effect of blue light on g(m). However, the residual reduction of g(m) under blue light was real and significant, although it did not appear to limit the chloroplast CO(2) concentration and, consequently, photosynthesis. Reduction of g(m) might be caused by chloroplast movement to avoid photodamage, in turn affecting the chloroplast surface exposed to intercellular spaces. However, g(m) reduction occurred immediately after exposure to blue light and was complete after less than 3 min, whereas chloroplast relocation was expected to occur more slowly. In addition, fast g(m) reduction was also observed after inhibiting chloroplast movement by cytochalasin. It is therefore concluded that g(m) reduction under blue light is unlikely to be caused by chloroplast movement only, and must be elicited by other, as yet unknown, factors.