Clonal and oligoclonal populations of gammadelta T cells, with respect to the expression of T cell receptors for antigen (Tcr), have been shown to localize in normal and inflamed tissues. The mechanisms responsible for the tissue-selective accumulation of these subsets are still not known. gammadelta T cells are the predominant T cell subset in newborn calves, making this animal a useful model to study these cells. However, molecular markers defining tissue-specific bovine ydelta T cell subsets are only now being developed. In this report, we describe three new anti-bovine gammadelta Tcr mAbs: GD3.8, GD197 and GD3.1, which provide useful tools to study these cells. GD3.8 recognized virtually all gammadelta T cells in the blood; whereas GD3.1 and GD197 recognized mutually exclusive as well as overlapping subsets. Using these three mAbs, four separate subsets of gammadelta T cells were defined: subset 1 (GD3.8+, GD3.1+, GD197-); subset 2 (GD3.8+, GD3.1-, GD197+); subset 3 (GD3.8+, GD3.1+, GD197+); and subset 4 (GD3.8+, GD3.1-, GD197-). Subset 4 constituted a minor population in the blood; however, it predominated in the spleen and, in some cases, represented a 300% increase over blood levels. The percentage of GD3.1-positive gammadelta T cells was found to be increased in experimentally inflamed lymph nodes, suggesting that subset 1 cells may be preferentially retained in or recruited to sites of inflammation. Some subset 4 cells also exhibited a decreased ability to respond to PHA. These studies demonstrate that bovine gammadelta T cell, Tcr-defined subsets, exhibit unique accumulation and activation characteristics that may provide clues to their function and regulation.