L-Glutamine is the most abundant free amino acid of the human body and is essential for the culture of many cell types. Clinically, reduction of glutamine by administration of glutaminase or the use of glutamine analogs is a common therapy for patients with acute lymphocytic leukemia. In the current study, we investigated the influence of glutamine concentrations on the human myelomonocytic cell line U937. Decreasing the glutamine concentration evoked a reduction in DNA synthesis (R2 = 0.9885, P < 0.0001), increased cell volume (P < 0.01) and the cytoplasm/nuclear ratio, and enhanced the development of vacuoles but did not influence cell viability. Culturing cells in reduced concentrations of glutamine augmented the percentage of cells expressing CD64 (Fc receptor for IgG/FcgammaRI, P < 0.01), CD11b (complement receptor type 3/CR3, P < 0.001) and CD71 (transferrin receptor, P < 0.05). The percentage of U937 cells expressing CD23 (low affinity receptor for IgE/FcepsilonRII) was increased at low concentrations of glutamine at both the protein (P < 0.01) and mRNA levels. The percentage of U937 cells phagocytizing opsonized E. coli (P < 0.001) or latex particles (P < 0.001) was enhanced by lowering the glutamine concentration. In conclusion, reducing glutamine concentration causes differentiation of the cell line U937 along the monocytic pathway. These effects may indicate a mechanistic basis for prior published evidence that glutaminase and glutamine antagonists are effective anti-tumor agents.