In a previous study thymocytes from young mice less than 3 weeks old, especially those of NK low reactive genotypes, were found sensitive for NK-mediated lysis. This phenomenon has been further explored in the present study in which the NK-sensitive thymocyte population of young strain A/Sn mice has been characterized. Injection of suckling A/Sn mice with cortisone drastically decreased sensitivity of the remaining medullary thymus cells for NK lysis, whereas sensitivity for lysis by anti-H-2-educated CTL blasts was enhanced. The NK-sensitive population repopulated the thymus after cortisone treatment 2 to 4 days earlier than the majority of the thymocytes. In vitro treatment of thymocytes with fresh rabbit or guinea pig serum, known to kill cortical thymocytes, also abolished the NK sensitivity, while sensitivity for allospecific CTL was increased. Depletion of thymocytes bearing receptors for the peanut agglutinin (PNA) strongly reduced the sensitivity of the remaining thymocytes for NK lysis, as well as for lysis by guinea pig and rabbit serum. Fractionation of thymocytes on a 1 × G velocity sedimentation gradient revealed a distinct sensitive peak sedimenting approximately 5 mm/hr whereas the majority of the thymocytes sedimented at the 3 to 4 mm/hr region. Morphologic studies confirmed that the majority of the NK-sensitive cells were large and rapidly dividing, indicating also by the high spontaneous incorporation of 3H thymidine into these 1 × G fractions. Free-flow electrophoresis analysis revealed that the NK-sensitive thymocytes distributed between the majority of the thymocytes and the rapidly migrating, more negatively charged medullary thymocyte. Taken together, these results show that the NK sensitive “natural” target is a minor population of the thymus, clearly distinguishable from the majority of the cortical as well as medullary thymocytes.