Minimum (F0), maximum (Fm), and variable (Fv = Fm - F0) fluorescence and photosynthetic efficiency (Fv/Fm) of phytoplankton were measured for several depths and under laboratory conditions during two seasons (June and October of 2000) in Lake Guatavita, Colombia. When the lake was stratified (June) the surface algae were photoinhibited and photosynthetic efficiency was very low, especially in the hypolimnion. In October, when the lake was circulating, efficiency was higher throughout the water column. Laboratory samples exhibited a gradual decline in efficiency over time, but with higher values in October. Significant differences between June and October samples were observed both in the laboratory and the field. Except for hypolimnetic samples for June, there were no significant differences between field observations and laboratory measures. When the lake was stratified, the concentration of chlorophyll-a was high in the hypolimnion, but the efficiency was low. During circulation, the concentration of chlorophyll-a was low but the efficiency was higher. The results demonstrate that the photosynthetic efficiency of the phytoplankton is independent of biomass and that in addition to biomass, other factors, such as availability of nutrients and light could affect photosynthetic efficiency.