Experimental investigations of ozone (O3) effects on plants have commonly used short, acute [O3] exposure (>100 ppb, on the order of hours), while in field crops damage is more likely caused by chronic exposure (<100 ppb, on the order of weeks). How different are the O3 effects induced by these two fumigation regimes? The leaf-level photosynthetic response of soybean to acute [O3] (400 ppb, 6 h) and chronic [O3] (90 ppb, 8 h d−1, 28 d) was contrasted via simultaneous in vivo measurements of chlorophyll a fluorescence imaging (CFI) and gas exchange. Both exposure regimes lowered leaf photosynthetic CO2 uptake about 40% and photosystem II (PSII) efficiency (Fq′/Fm′) by 20% compared with controls, but this decrease was far more spatially heterogeneous in the acute treatment. Decline in Fq′/Fm′ in the acute treatment resulted equally from decreases in the maximum efficiency of PSII (Fv′/Fm′) and the proportion of open PSII centres (Fq′/Fv′), but in the chronic treatment decline in Fq′/Fm′ resulted only from decrease in Fq′/Fv′. Findings suggest that acute and chronic [O3] exposures do not induce identical mechanisms of O3 damage within the leaf, and using one fumigation method alone is not sufficient for understanding the full range of mechanisms of O3 damage to photosynthetic production in the field.