Gene expression profiles were compared in circulating bovine GD3.5+ (CD8-) and GD3.5- (predominantly CD8+) gammadelta T cells using serial analysis of gene expression (SAGE). Approximately 20,000 SAGE tags were generated from each library. A comparison of the two libraries demonstrated 297 and 173 tags representing genes with 5-fold differential expression in GD3.5+ and GD3.5- gammadelta T cells, respectively. Consistent with their localization into sites of inflammation, GD3.5+ gammadelta T cells appeared transcriptionally and translationally more active than GD3.5- gammadelta cells. GD3.5- gammadelta T cells demonstrated higher expression of the cell proliferation inhibitor BAP 37, which was associated with their less activated gene expression phenotype. The immune regulatory and apoptosis-inducing molecule, galectin-1, was identified as a highly abundant molecule and was higher in GD3.5+gammadelta T cells. Surface molecules attributed to myeloid cells, such as CD14, CD68, and scavenger receptor-1, were identified in both populations. Furthermore, expression of B lymphocyte-induced maturation protein, a master regulator of B cell and myeloid cell differentiation, was identified by SAGE analysis and was confirmed at the RNA level to be selectively expressed in gammadelta T cells vs alphabeta T cells. These results provide new insights into the inherent differences between circulating gammadelta T cell subsets.