Subpopulations of rat T lymphocytes described in this paper are defined by their susceptibility to the selective cytotoxic effects of normal guinea pig serum (GPS). These cells were present in relatively large proportions in the bone marrow and thymus and in very small percentages in the spleen and lymph nodes. Tissue distribution of GPS-sensitive lymphocytes was similar in three inbred strains and, therefore, was characteristic of the species rather than a single strain. Adherent peritoneal cells were largely insensitive to GPS. The partial cytotoxicity of GPS on rat lymphoid cells contrasted with the indiscriminate killing capacity of normal sera from humans, rabbits, and chickens, which affected over 98% of the cells regardless of organ location. Variations of the number of cells used in otherwise identical GPS-cytotoxicity assays did not significantly alter the proportion of either thymus or bone marrow cells killed. This suggests a selective sensitivity by discrete subclasses of lymphocytes present in these organs. GPS-sensitive lymphocytes occurred in rat bone marrow from birth and constituted 30 to 50% of the nucleated cells. The percentage of these cells decreased after 5 weeks of age to 15 to 30%, with most value being around 20%. Cells susceptible to GPS cytotoxicity were absent in the thymus of 35-day-old or younger rats but were readily detectable in 8-week-old animals, suggesting that GPS-reactivity defines a thymus cell surface-differentiation component. The subset of the thymus cell expressing this marker was essential for responsiveness to either PHA or Con A. Prior killing of bone marrow cells with GPS left no cells to be killed specifically by rabbit anti-rat Thy 1 IgG plus complement (C) free of nonspecific cytotoxicity. Initial killing of bone marrow cells with anti-Thy 1 and C consistently eliminated the GPS-sensitive cells. These results indicate that in the rat bone marrow, unlike in the thymus, GPS-sensitive and Thy 1-bearing lymphocytes constitute the same cell subpopulation. GPS is capable of “recognizing” and killing subpopulations of rat T lymphocytes, a property that can be used to characterize further these cells on a functional basis.