Antibodies to GM1 ganglioside were used to study murine lymphocyte populations. In A, AKR, and BALB/c mice, anti-GM1 reacts with thymocytes and peripheral T cells. This reactivity of anti-GM1, studied by immunofluorescence, is independent of Thy-1 type and appears to be related to the reactivity of cross-reacting antibodies to asialo GM1 and GD1b, rather than GM1 itself. In addition, a subpopulation of lymphocytes reacting with anti-GM1 and anti-immunoglobulin has been found in approximately 26% of the peripheral lymphocytes of C3H mice, nude mice, and nude heterozygotes. This subpopulation is found in small numbers in A, AKR, and BALB/c mice. These studies demonstrate that antibodies to a chemically defined antigen can be used to identify T cells in many strains of mice and may delineate previously unrecognized lymphocyte subpopulations.