We describe the theory and practice of estimating photosynthetic rates from light‐stimulated changes in the quantum yield of chlorophyll fluorescence. By means of a pump‐and‐probe fluorescence technique, where weak probe flashes are used to measure the change in the quantum yield of fluorescence induced by the strong pump flash, it is possible to derive the absolute absorption cross sections for photosystem 2, the quantum yield for photochemistry, and the maximum rate of photosynthetic electron transport at light saturation. In conjunction with a semiempirical biophysical model of photosynthesis, these parameters can be used to calculate the instantaneous rate of gross photosynthesis in situ under ambient irradiance. A profiling pump‐and‐probe fluorometer was constructed and interfaced with a CTD, and vertical profiles of variable fluorescence were obtained on four cruises in the northwest Atlantic Ocean. The derived photosynthetic rates were compared with concurrent estimates of production based on radiocarbon uptake. The correlation coefficient between the two estimates of primary production, normalized to Chl a, was 0.86; linear regression analysis yielded a slope of 1.06. There is a 3‐4‐fold range in the maximum change in the quantum yields of photochemistry and absorption cross‐sections in natural phytoplankton communities. Uncertainties in the pump‐and‐probe‐derived estimates of photosynthesis are primarily due to temporal mismatches between instantaneous and time‐integrated measures of production and in biological variability in the ratio of the number of PS2 reaction centers to total Chl a.