Murine thymus cells were maintained in vitro with supernatant-factors derived from Concanavalin-A-stimulated spleen cells. After an initial phase of vigorous proliferation, large granular cells (GC), which were not observed in fresh thymus cell preparations, appeared in these cultures. GC, derived from C3Hf/Tif-, BALB/c-, and C57BL/10-thymus cultures, could be slowly expanded and have been maintained as increasingly homogeneous (oligoclonal) lines for up to six months. During this time, other types of thymus cells died or were diluted out. Thymic GC differ functionally and histochemically from macrophages and mast cells. They do not phagocytize zymosan particles, bind opsonized SRBC, express nonspecific esterases or contain detectable amounts of histamine. GC share many features with natural killer (NK) cells and large granular lymphocytes (LGL). One morphologically representative line (C3Hf/Tif) had the following surface phenotype: Thy-1+, Lyt-1-, Lyt-2-, H-2K+, I-A-, asialo Gm1+. GC bind peanut agglutinin (PNA) on their surface and contain azurophilic granules. These cytoplasmic granules are considerably larger than those in LGL. Cells of a GC line derived from mouse strain C3Hf/Tif (H-2k) lysed the NK-sensitive YAC-1 (H-2a) and EL-4 (H-2b), but not the NK-insensitive P815 cells.