A noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos is described. A linear relationship was found between fluorescence intensity of NADH produced and glutamine concentration (R2 = 0.985). Single embryos were placed in 20 nl drops of medium containing 0.5 mM glutamine, and the medium was sampled after 2 hr incubation at 37 degrees C. Changes in net glutamine uptake were determined in one-cell, two-cell, and eight-cell embryos and blastocysts incubated in medium containing no energy substrates (glucose, pyruvate, and lactate). The median glutamine uptake increased significantly from 0.480 and 0.270 pmoles/embryo/2 hr at the one-cell and two-cell stages, respectively, to 1.610 pmoles/embryo/2 hr at the blastocyst stage. Mean glutamine uptake was compared in the presence or absence of energy substrates at several developmental stages. A highly significant reduction of glutamine uptake in the presence of substrates was observed at the one-cell and two-cell stages of development. At the eight-cell stage, glutamine uptake was only marginally reduced in the presence of substrates, and no effect was found at the blastocyst stage. These data may partially explain the beneficial effect of glutamine on the culture of early mouse embryos through the two-cell block of development.